JP2022547786A - Hantavirus antigenic composition - Google Patents

Abstract

The present invention provides a viral or bacterial vector comprising a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof and capable of inducing a protective immune response in a subject. The invention also provides compositions and uses of vectors in methods of medical treatment.

Description

The present invention relates to viral and bacterial vectors containing hantavirus antigens and their use in immunogenic and antigenic compositions. The invention also relates to the prophylactic use of said composition. The invention also relates to immunogens for use in producing therapeutic antibodies and methods for producing said immunogens.

Hantaviruses are emerging zoonotic viruses with worldwide distribution. There are numerous strains of hantavirus, which are broadly classified into three serotypes. Hantavirus is the causative agent of hantavirus pulmonary syndrome (HPS), a serious human respiratory disease that is typically fatal in 36% of cases, and 50% during some outbreaks. Mortality has been recorded. It is also the causative agent of hemorrhagic fever with renal syndrome (HFRS), a clinically similar group of diseases that can be fatal in up to 15% of cases. Hantaviruses are transmitted to humans, typically through exposure to aerosolized bodily fluids or faeces of infected small mammals, typically rodents. Human-to-human transmission has also been reported.

According to the Centers for Disease Control and Prevention (CDC), symptoms associated with hantavirus infection include fever, headache, muscle aches, and severe breathing difficulties. Symptoms associated with HPS can also include fatigue, chills, dizziness, dry cough, nausea, vomiting, and other gastrointestinal symptoms, as well as malaise, diarrhea, mild headaches, joint pain, back pain, and abdominal pain. Symptoms associated with HFRS include severe headaches, back and abdominal pain, fever, chills, nausea, blurred vision, flushing, eye irritation or redness, and rash. Symptoms after HFRS can include hypotension, acute shock, vascular leakage, and acute renal failure, which can cause severe fluid overload.

There is currently no licensed vaccine against hantavirus. According to the CDC, there is no definite treatment or cure for hantavirus infection, HPS or HFRS. Patients with HPS are admitted to intensive care units and treated with intubation and oxygen therapy to support the patient during severe respiratory distress. Successful treatment of HPS depends on the severity of dyspnea and early detection of infection. Treatment of HFRS can include control of the patient's fluid and electrolyte levels, oxygen and blood pressure levels, dialysis to correct severe fluid overload, and treatment of any secondary infections. The antiviral drug ribavirin has been shown to reduce morbidity and mortality when used very early in the clinical course of HFRS. However, no benefit of ribavirin has been found for HPS patients.

Therefore, there is an important need for a protective vaccine against hantavirus infection. Additional therapeutic agents for the prevention, treatment and control of hantavirus infections are also urgently needed.

The present invention provides viral and bacterial vectors encoding hantavirus nucleoprotein (NP) or antigenic fragments thereof, along with corresponding compositions and uses of said vectors and compositions in the prevention and treatment of hantavirus infections. addresses one or more of the above problems.

The vectors and compositions of the invention can stimulate (ie, induce) an immune response in an individual (ie, subject) against hantavirus, providing improved immunogenicity and efficacy.

In one aspect, the invention provides a viral or bacterial vector comprising a nucleic acid sequence encoding hantavirus NP or an antigenic fragment thereof and capable of inducing an immune response in an individual. The inventors have demonstrated that by using a viral or bacterial vector to deliver a nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) to a subject, a highly effective immune response to hantavirus can be produced in an individual. We found that it can be generated in

In preferred embodiments, the vectors of the invention are viral vectors.

Hantaviruses are a genus of enveloped, single-stranded, tripartite, negative-strand RNA viruses belonging to the Bunyaviridae family. More than 20 hantavirus strains that are pathogenic to humans have been described, each strain being matched to a single rodent species. Hantavirus strains are broadly classified as either Old World or New World. Old World strains include Seoul virus (“SEOV”; global distribution), Puumala virus (primarily European distribution), Hantan virus (“HNT”; primarily Asian distribution) and Dobrava virus (primarily European distribution) and is typically associated with the cause of HFRS. New World strains include Shin Nombre virus (mainly distributed in North America) and Andes virus (mainly distributed in Latin America) and are typically associated with HPS.

The hantavirus genome consists of three single-stranded RNA segments called small (S), medium (M), and large (L). The S segment is 1-3 kb and encodes the nucleocapsid protein (NP). The M segment is 3.2-4.9 kb and encodes glycoproteins (GP), Gn and Gc. The L segment is 6.8-12 kb and encodes the viral RNA-dependent RNA polymerase.

Hantavirus glycoproteins, Gn and Gc, play an important role in the infection of target cells through interaction with specific entry receptors such as integrins. Hantavirus NP forms a ribonucleoprotein complex with the viral polymerase and plays multiple roles in viral propagation. NP has also been reported to play a role in enhancing translation of viral RNA by host cells, downregulating apoptosis, inhibiting interferon signaling responses, and blocking TNFα-induced activation of NF-κB.

Seoul virus can be used as a reference hantavirus strain. GenBank Accession No. KM948598.1 provides a reference nucleic acid sequence for Hantavirus NP (see SEQ ID NO: 1) and a reference polypeptide sequence for Hantavirus NP (SEQ ID NO: 4).
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The coding sequence of SEQ ID NO:1 corresponds to nucleic acid residues 43-1332 therein and is represented by SEQ ID NO:2.
ＡＴＧＧＣＡＡＣＴＡＴＧＧＡＡＧＡＡＡＴＣＣＡＧＡＧＡＧＡＡＡＴＣＡＧＴＧＣＧＣＡＣＧＡＧＧＧＧＣＡＧＣＴＴＧＴＡＡＴＡＧＣＡＣＧＣＣＡＧＡＡＧＧＴＣＡＡＧＧＡＴＧＣＡＧＡＡＡＡＧＣＡＧＴＡＴＧＡＧＡＡＧＧＡＴＣＣＴＧＡＴＧＡＣＣＴＡＡＡＴＡＡＧＡＧＧＧＣＡＣＴＧＣＡＴＧＡＴＣＧＧＧＡＧＡＧＴＧＴＣＧＣＡＧＣＴＴＣＡＡＴＡＣＡＡＴＣＡＡＡＡＡＴＴＧＡＴＧＡＡＴＴＧＡＡＧＣＧＣＣＡＡＣＴＴＧＣＴＧＡＣＡＧＧＡＴＴＧＣＡＧＣＡＧＧＧＡＡＧＡＡＣＡＴＣＧＧＧＣＡＡＧＡＣＣＧＧＧＡＴＣＣＴＡＣＡＧＧＧＧＴＡＧＡＧＣＣＧＧＧＴＧＡＴＣＡＴＣＴＣＡＡＧＧＡＡＡＧＡＴＣＡＧＣＡＣＴＡＡＧＣＴＡＣＧＧＧＡＡＴＡＣＡＣＴＧＧＡＣＣＴＧＡＡＴＡＧＣＣＴＴＧＡＣＡＴＴＧＡＴＧＡＡＣＣＴＡＣＡＧＧＡＣＡＧＡＣＡＧＣＴＧＡＴＴＧＧＴＴＧＡＣＣＡＴＡＡＴＴＧＴＣＴＡＴＴＴＧＡＣＡＴＣＡＴＴＣＧＴＧＧＴＣＣＣＧＡＴＣＡＴＣＴＴＧＡＡＧＧＣＡＣＴＧＴＡＣＡＴＧＴＴＧＡＣＡＡＣＡＡＧＡＧＧＣＡＧＧＣＡＧＡＣＴＴＣＡＡＡＧＧＡＣＡＡＣＡＡＧＧＧＡＡＴＧＡＧＧＡＴＣＡＧＡＴＴＣＡＡＧＧＡＴＧＡＣＡＧＣＴＣＡＴＡＴＧＡＡＧＡＴＧＴＣＡＡＴＧＧＡＡＴＣＡＧＡＡＡＧＣＣＣＡＡＡＣＡＴＣＴＧＴＡＴＧＴＧＴＣＡＡＴＧＣＣＡＡＡＣＧＣＣＣＡＡＴＣＡＡＧＣＡＴＧＡＡＧＧＣＴＧＡＡＧＡＧＡＴＡＡＣＡＣＣＴＧＧＡＡＧＡＴＴＣＣＧＣＡＣＴＧＣＡＧＴＡＴＧＴＧＧＧＣＴＡＴＡＣＣＣＴＧＣＡＣＡＧＡＴＡＡＡＧＧＣＡＡＧＧＡＡＣＡＴＧＧＴＡＡＧＣＣＣＴＧＴＣＡＴＧＡＧＴＧＴＡＧＴＴＧＧＧＴＴＴＴＴＧＧＣＡＣＴＧＧＣＡＡＡＡＧＡＣＴＧＧＡＣＡＴＣＴＡＧＡＡＴＴＧＡＡＧＡＡＴＧＧＣＴＴＧＧＴＧＣＡＣＣＣＴＧＣＡＡＧＴＴＣＡＴＧＧＣＡＧＡＧＴＣＴＣＣＣＡＴＴＧＣＣＧＧＧＡＧＣＴＴＡＴＣＴＧＧＧＡＡＴＣＣＴＧＴＧＡＡＴＣＧＴＧＡＴＴＡＴＡＴＣＡＧＡＣＡＧＡＧＡＣＡＡＧＧＴＧＣＡＣＴＴＧＣＡＧＧＧＡＴＧＧＡＧＣＣＡＡＡＡＧＡＡＴＴＴＣＡＡＧＣＴＣＴＣＡＧＧＣＡＡＣＡＴＴＣＡＡＡＧＧＡＴＧＣＴＧＧＡＴＧＴＡＣＡＣＴＧＧＴＴＧＡＡＣＡＴＡＴＴＧＡＧＴＣＡＣＣＡＴＣＡＴＣＡＡＴＡＴＧＧＧＴＡＴＴＴＧＣＴＧＧＧＧＣＣＣＣＴＧＡＴＡＧＧＴＧＣＣＣＡＣＣＧＡＣＡＴＧＣＣＴＧＴＴＴＧＴＴＧＧＡＧＧＧＡＴＧＧＣＴＧＡＧＴＴＡＧＧＴＧＣＴＴＴＣＴＴＴＴＣＴＡ ＴＡＣＴＴＣＡＧＧＡＴＡＴＧＡＧＧＡＡＣＡＣＡＡＴＣＡＴＧＧＣＴＴＣＡＡＡＧＡＣＴＧＴＧＧＧＡＡＣＡＧＣＴＧＡＴＧＡＡＡＡＧＣＴＴＣＧＡＡＡＧＡＡＧＴＣＡＴＣＡＴＴＣＴＡＴＣＡＡＴＣＡＴＡＣＣＴＣＡＧＡＣＧＣＡＣＡＣＡＡＴＣＡＡＴＧＧＧＡＡＴＡＣＡＡＣＴＧＧＡＣＣＡＧＡＧＧＡＴＡＡＴＴＧＴＴＡＴＧＴＴＴＡＴＧＧＴＴＧＣＣＴＧＧＧＧＡＡＡＧＧＡＧＧＣＡＧＴＧＧＡＣＡＡＣＴＴＴＣＡＴＣＴＣＧＧＴＧＡＴＧＡＣＡＴＧＧＡＴＣＣＡＧＡＧＣＴＴＣＧＣＡＧＣＣＴＧＧＣＴＣＡＧＡＴＣＣＴＧＡＴＴＧＡＣＣＡＧＡＡＡＧＴＧＡＡＧＧＡＡＡＴＣＴＣＡＡＡＣＣＡＧＧＡＡＣＣＴＡＴＧＡＡＡＴＴＡ（配列番号２）

The inventors have generated a nucleic acid sequence encoding hantavirus NP that has been optimized for expression in Homo sapiens (see SEQ ID NO:3):
ＡＴＧＧＣＣＡＣＡＡＴＧＧＡＡＧＡＧＡＴＣＣＡＧＡＧＡＧＡＧＡＴＣＡＧＣＧＣＣＣＡＣＧＡＧＧＧＡＣＡＧＣＴＧＧＴＴＡＴＣＧＣＣＡＧＡＣＡＧＡＡＡＧＴＧＡＡＧＧＡＣＧＣＣＧＡＧＡＡＧＣＡＧＴＡＣＧＡＧＡＡＧＧＡＣＣＣＣＧＡＣＧＡＴＣＴＧＡＡＣＡＡＧＡＧＡＧＣＣＣＴＧＣＡＣＧＡＣＡＧＡＧＡＡＡＧＣＧＴＧＧＣＣＧＣＣＴＣＴＡＴＣＣＡＧＡＧＣＡＡＧＡＴＣＧＡＴＧＡＧＣＴＧＡＡＧＡＧＡＣＡＧＣＴＧＧＣＣＧＡＣＡＧＡＡＴＣＧＣＣＧＣＴＧＧＣＡＡＧＡＡＴＡＴＴＧＧＣＣＡＧＧＡＣＡＧＡＧＡＴＣＣＣＡＣＡＧＧＣＧＴＧＧＡＡＣＣＴＧＧＣＧＡＴＣＡＣＣＴＧＡＡＡＧＡＧＡＧＡＡＧＣＧＣＣＣＴＧＴＣＣＴＡＴＧＧＣＡＡＣＡＣＣＣＴＧＧＡＣＣＴＧＡＡＣＡＧＣＣＴＧＧＡＣＡＴＴＧＡＴＧＡＧＣＣＴＡＣＣＧＧＣＣＡＧＡＣＡＧＣＣＧＡＣＴＧＧＣＴＧＡＣＡＡＴＣＡＴＴＧＴＧＴＡＣＣＴＧＡＣＣＡＧＣＴＴＣＧＴＧＧＴＣＣＣＣＡＴＣＡＴＣＣＴＧＡＡＧＧＣＣＣＴＧＴＡＣＡＴＧＣＴＧＡＣＣＡＣＣＡＧＡＧＧＣＡＧＡＣＡＧＡＣＣＡＧＣＡＡＧＧＡＣＡＡＣＡＡＧＧＧＣＡＴＧＡＧＡＡＴＣＣＧＧＴＴＣＡＡＧＧＡＴＧＡＣＡＧＣＡＧＣＴＡＣＧＡＧＧＡＣＧＴＧＡＡＣＧＧＣＡＴＴＡＧＡＡＡＧＣＣＣＡＡＧＣＡＣＣＴＧＴＡＣＧＴＧＴＣＣＡＴＧＣＣＴＡＡＣＧＣＴＣＡＧＡＧＣＡＧＣＡＴＧＡＡＧＧＣＣＧＡＧＧＡＡＡＴＣＡＣＣＣＣＴＧＧＣＡＧＡＴＴＣＡＧＡＡＣＡＧＣＣＧＴＧＴＧＣＧＧＡＣＴＧＴＡＣＣＣＣＧＣＴＣＡＧＡＴＣＡＡＧＧＣＣＡＧＡＡＡＣＡＴＧＧＴＧＴＣＣＣＣＡＧＴＧＡＴＧＡＧＣＧＴＣＧＴＧＧＧＡＴＴＴＣＴＧＧＣＣＣＴＧＧＣＴＡＡＧＧＡＣＴＧＧＡＣＣＡＧＣＡＧＧＡＴＴＧＡＧＧＡＡＴＧＧＣＴＧＧＧＡＧＣＣＣＣＴＴＧＣＡＡＧＴＴＴＡＴＧＧＣＣＧＡＧＴＣＴＣＣＴＡＴＣＧＣＣＧＧＣＡＧＣＣＴＧＴＣＴＧＧＣＡＡＣＣＣＣＧＴＧＡＡＴＡＧＡＧＡＣＴＡＣＡＴＣＡＧＡＣＡＧＡＧＧＣＡＧＧＧＣＧＣＴＣＴＧＧＣＣＧＧＡＡＴＧＧＡＡＣＣＣＡＡＡＧＡＡＴＴＴＣＡＧＧＣＣＣＴＧＣＧＧＣＡＧＣＡＣＴＣＴＡＡＧＧＡＴＧＣＣＧＧＡＴＧＴＡＣＣＣＴＧＧＴＧＧＡＡＣＡＣＡＴＴＧＡＧＡＧＣＣＣＣＡＧＣＡＧＣＡＴＣＴＧＧＧＴＴＴＴＣＧＣＴＧＧＣＧＣＴＣＣＴＧＡＴＡＧＡＴＧＣＣＣＴＣＣＴＡＣＣＴＧＴＣＴＧＴＴＴＧＴＴＧＧＣＧＧＡＡＴＧＧＣＣＧＡＧＣＴＧＧＧＣＧＣＣＴＴＣＴＴＴＡＧＣＡ ＴＴＣＴＧＣＡＧＧＡＣＡＴＧＣＧＧＡＡＴＡＣＣＡＴＣＡＴＧＧＣＣＡＧＣＡＡＧＡＣＣＧＴＧＧＧＣＡＣＣＧＣＣＧＡＴＧＡＧＡＡＧＣＴＧＡＧＡＡＡＧＡＡＧＴＣＣＡＧＣＴＴＣＴＡＣＣＡＧＡＧＣＴＡＣＣＴＧＣＧＧＡＧＡＡＣＣＣＡＧＡＧＣＡＴＧＧＧＣＡＴＴＣＡＧＣＴＧＧＡＣＣＡＧＡＧＡＡＴＣＡＴＣＧＴＧＡＴＧＴＴＣＡＴＧＧＴＧＧＣＣＴＧＧＧＧＣＡＡＡＧＡＡＧＣＣＧＴＧＧＡＣＡＡＴＴＴＴＣＡＣＣＴＧＧＧＣＧＡＣＧＡＣＡＴＧＧＡＣＣＣＣＧＡＧＣＴＧＡＧＡＴＣＴＣＴＧＧＣＣＣＡＧＡＴＣＣＴＧＡＴＣＧＡＣＣＡＧＡＡＡＧＴＣＡＡＡＧＡＧＡＴＣＴＣＣＡＡＴＣＡＡＧＡＧＣＣＣＡＴＧＡＡＧＣＴＧ（配列番号３）。

Translation of the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:3 yields the hantavirus NP polypeptide sequence, which is represented by (SEQ ID NO:4):
ＭＡＴＭＥＥＩＱＲＥＩＳＡＨＥＧＱＬＶＩＡＲＱＫＶＫＤＡＥＫＱＹＥＫＤＰＤＤＬＮＫＲＡＬＨＤＲＥＳＶＡＡＳＩＱＳＫＩＤＥＬＫＲＱＬＡＤＲＩＡＡＧＫＮＩＧＱＤＲＤＰＴＧＶＥＰＧＤＨＬＫＥＲＳＡＬＳＹＧＮＴＬＤＬＮＳＬＤＩＤＥＰＴＧＱＴＡＤＷＬＴＩＩＶＹＬＴＳＦＶＶＰＩＩＬＫＡＬＹＭＬＴＴＲＧＲＱＴＳＫＤＮＫＧＭＲＩＲＦＫＤＤＳＳＹＥＤＶＮＧＩＲＫＰＫＨＬＹＶＳＭＰＮＡＱＳＳＭＫＡＥＥＩＴＰＧＲＦＲＴＡＶＣＧＬＹＰＡＱＩＫＡＲＮＭＶＳＰＶＭＳＶＶＧＦＬＡＬＡＫＤＷＴＳＲＩＥＥＷＬＧＡＰＣＫＦＭＡＥＳＰＩＡＧＳＬＳＧＮＰＶＮＲＤＹＩＲＱＲＱＧＡＬＡＧＭＥＰＫＥＦＱＡＬＲＱＨＳＫＤＡＧＣＴＬＶＥＨＩＥＳＰＳＳＩＷＶＦＡＧＡＰＤＲＣＰＰＴＣＬＦＶＧＧＭＡＥＬＧＡＦＦＳＩＬＱＤＭＲＮＴＩＭＡＳＫＴＶＧＴＡＤＥＫＬＲＫＫＳＳＦＹＱＳＹＬＲＲＴＱＳＭＧＩＱＬＤＱＲＩＩＶＭＦＭＶＡＷＧＫＥＡＶＤＮＦＨＬＧＤＤＭＤＰＥＬＲＳＬＡＱＩＬＩＤＱＫＶＫＥＩＳＮＱＥＰＭＫＬ（配列番号４）。

Hantavirus can be used as a reference hantavirus strain. GenBank Accession No. KC570390.1 provides a reference nucleic acid sequence for Hantavirus NP (see SEQ ID NO:5) and a reference polypeptide sequence for Hantavirus NP (see SEQ ID NO:7).
ＴＡＧＴＡＧＴＡＧＡＣＴＣＣＣＴＡＡＡＧＡＧＣＴＡＣＴＡＧＡＡＣＡＡＣＧＡＴＧＧＣＡＡＣＴＡＴＧＧＡＧＧＡＡＴＴＧＣＡＧＡＧＧＧＡＡＡＴＣＡＡＴＧＣＣＣＡＴＧＡＧＧＧＴＣＡＡＣＴＧＧＴＧＡＴＡＧＣＣＡＧＧＣＡＧＡＡＧＧＴＧＡＧＧＧＡＴＧＣＡＧＡＡＡＡＧＣＡＧＴＡＴＧＡＡＡＡＧＧＡＴＣＣＡＧＡＴＧＡＧＴＴＡＡＡＣＡＡＧＡＧＡＧＣＡＴＴＧＡＣＡＧＡＴＣＧＡＧＡＧＧＧＴＧＴＴＧＣＡＧＴＡＴＣＣＡＴＴＣＡＡＧＣＡＡＡＧＡＴＴＧＡＴＧＡＧＴＴＡＡＡＧＡＧＧＣＡＡＴＴＧＧＣＡＧＡＴＡＧＧＡＴＴＧＣＡＡＣＣＧＧＧＡＡＧＡＡＣＣＴＴＧＧＡＡＡＧＧＡＡＣＡＡＧＡＣＣＣＡＡＣＡＧＧＧＧＴＡＧＡＡＣＣＴＧＧＡＧＡＴＣＡＴＣＴＧＡＡＡＧＡＧＡＧＡＴＣＡＡＴＧＣＴＣＡＧＴＴＡＴＧＧＡＡＡＴＧＴＴＣＴＴＧＡＣＴＴＡＡＡＣＣＡＣＣＴＧＧＡＴＡＴＴＧＡＴＧＡＧＣＣＡＡＣＡＧＧＡＣＡＧＡＣＡＧＣＡＧＡＣＴＧＧＣＴＧＧＧＣＡＴＴＧＴＴＡＴＣＴＡＴＣＴＣＡＣＡＴＣＣＴＴＴＧＴＴＧＴＣＣＣＧＡＴＡＣＴＴＣＴＧＡＡＡＧＣＣＣＴＧＴＡＣＡＴＧＴＴＡＡＣＡＡＣＡＡＧＡＧＧＧＡＧＧＣＡＧＡＣＣＡＣＣＡＡＧＧＡＣＡＡＴＡＡＡＧＧＡＡＣＴＣＧＧＡＴＴＣＧＡＴＴＣＡＡＧＧＡＴＧＡＴＡＧＣＴＣＣＴＴＣＧＡＧＧＡＴＧＴＣＡＡＴＧＧＣＡＴＴＣＧＧＡＡＧＣＣＧＡＡＡＣＡＴＣＴＡＴＡＴＧＴＧＴＣＣＴＴＡＣＣＡＡＡＴＧＣＡＣＡＧＴＣＡＡＧＴＡＴＧＡＡＡＧＣＡＧＡＡＧＡＧＡＴＴＡＣＡＣＣＴＧＧＴＡＧＡＴＡＴＡＧＡＡＣＡＧＣＡＡＴＴＴＧＴＧＧＡＣＴＴＴＡＣＣＣＴＧＣＡＣＡＡＡＴＴＡＡＧＧＣＡＡＧＡＣＡＧＡＴＧＡＴＴＡＧＴＣＣＡＧＴＣＡＴＧＡＧＴＧＴＡＡＴＣＧＧＡＴＴＣＴＴＧＧＣＴＴＴＧＧＣＡＡＡＡＧＡＴＴＧＧＡＧＴＧＡＣＣＧＣＡＴＴＧＡＧＣＡＧＴＧＧＴＴＡＡＧＴＧＡＡＣＣＧＴＧＴＡＡＧＣＴＴＣＴＴＣＣＡＧＡＴＡＣＡＧＣＡＧＣＡＧＴＴＡＧＣＣＴＴＣＴＴＧＧＴＧＧＴＣＣＴＧＣＡＡＣＣＡＡＣＡＧＧＧＡＣＴＡＴＴＴＡＣＧＧＣＡＧＣＧＡＣＡＡＧＴＡＧＣＡＴＴＧＧＧＣＡＡＣＡＴＧＧＡＡＡＣＡＡＡＡＧＡＧＴＣＴＡＡＧＧＣＴＡＴＡＣＧＣＣＡＡＣＡＴＧＣＡＧＡＡＧＣＡＧＣＡＧＧＣＴＧＴＡＧＴＡＴＧＡＴＴＧＡＧＧＡＣＡＴＴＧＡＧＴＣＡＣＣＡＴＣＡＴＣＡＡＴＡＴＧＧＧＴＧＴＴＴＧＣＴＧＧＧＧＣＡＣＣＧＧＡＣＣＧＣＴＧＴＣＣＡＣＣＡＡＣＡＴＧＴＣＴＣＴＴＴＡ ＴＴＧＣＡＧＧＴＡＴＧＧＣＴＧＡＧＣＴＴＧＧＧＧＣＡＴＴＴＴＴＴＴＣＣＡＴＣＣＴＧＣＡＧＧＡＣＡＴＧＣＧＡＡＡＴＡＣＡＡＴＴＡＴＧＧＣＡＴＣＣＡＡＧＡＣＡＧＴＴＧＧＡＡＣＣＴＣＴＧＡＧＧＡＧＡＡＧＣＴＡＣＧＧＡＡＧＡＡＡＴＣＣＴＣＡＴＴＣＴＡＴＣＡＧＴＣＴＴＡＴＣＴＣＡＧＧＡＧＡＡＣＡＣＡＡＴＣＡＡＴＧＧＧＡＡＴＡＣＡＡＣＴＧＧＡＴＣＡＧＡＧＧＡＴＡＡＴＴＧＴＧＣＴＣＴＴＣＡＴＧＧＴＡＧＣＣＴＧＧＧＧＧＡＡＡＧＡＡＧＣＡＧＴＧＧＡＴＡＡＣＴＴＣＣＡＣＣＴＡＧＧＡＧＡＴＧＡＴＡＴＧＧＡＣＣＣＴＧＡＧＣＴＧＣＧＡＡＣＡＣＴＡＧＣＡＣＡＧＡＧＣＣＴＧＡＴＴＧＡＴＧＴＴＡＡＡＧＴＧＡＡＧＧＡＡＡＴＴＴＣＣＡＡＣＣＡＡＧＡＧＣＣＴＴＴＡＡＡＡＣＴＡＴＡＡＴＣＡＧＴＧＡＡＴＧＴＡＴＡＡＣＣＣＴＣＡＴＴＡＴＧＴＧＡＴＴＡＴＴＡＴＡＴＡＣＴＡＣＴＧＡＡＴＣＡＴＴＡＴＣＡＡＴＣＡＴＡＴＴＴＧＣＡＣＴＡＴＴＡＴＴＡＴＣＡＧＧＧＧＡＡＴＴＡＧＴＡＴＡＴＣＡＧＧＧＴＡＡＧＧＧＣＡＣＡＴＴＴＡＴＧＧＧＴＧＧＧＡＡＴＣＡＴＴＡＣＴＣＡＧＡＧＧＧＴＧＧＧＴＣＡＧＴＴＡＡＴＣＣＧＴＴＧＴＧＧＧＴＧＧＧＴＴＴＡＧＴＴＣＣＴＧＧＣＴＧＣＣＴＴＡＡＧＴＡＧＣＣＴＴＴＴＴＴＴＧＴＡＴＡＴＡＴＧＧＡＴＧＴＡＧＡＴＴＴＣＡＴＴＴＧＡＴＣＴＴＴＡＡＡＣＴＡＡＴＣＴＴＧＣＴＣＴＴＴＴＴＣＣＴＴＴＴＣＣＴＣＣＴＧＣＴＴＴＣＴＣＴＧＣＴＴＡＣＴＡＡＣＡＡＣＡＡＣＡＴＴＣＴＡＣＣＴＣＡＡＣＡＣＡＣＡＡＣＴＡＣＣＴＣＡＡＣＴＡＡＡＣＴＡＣＣＴＣＡＴＴＴＧＡＴＴＧＣＴＣＣＴＴＧＡＴＴＧＴＣＴＣＴＴＴＡＧＧＧＡＧＴＣＴＡＣＴＡＣＴＡ（配列番号５）。

The coding sequence of SEQ ID NO:5 corresponds to nucleic acid residues 37-1323 therein and is represented by SEQ ID NO:6.
ＡＴＧＧＣＡＡＣＴＡＴＧＧＡＧＧＡＡＴＴＧＣＡＧＡＧＧＧＡＡＡＴＣＡＡＴＧＣＣＣＡＴＧＡＧＧＧＴＣＡＡＣＴＧＧＴＧＡＴＡＧＣＣＡＧＧＣＡＧＡＡＧＧＴＧＡＧＧＧＡＴＧＣＡＧＡＡＡＡＧＣＡＧＴＡＴＧＡＡＡＡＧＧＡＴＣＣＡＧＡＴＧＡＧＴＴＡＡＡＣＡＡＧＡＧＡＧＣＡＴＴＧＡＣＡＧＡＴＣＧＡＧＡＧＧＧＴＧＴＴＧＣＡＧＴＡＴＣＣＡＴＴＣＡＡＧＣＡＡＡＧＡＴＴＧＡＴＧＡＧＴＴＡＡＡＧＡＧＧＣＡＡＴＴＧＧＣＡＧＡＴＡＧＧＡＴＴＧＣＡＡＣＣＧＧＧＡＡＧＡＡＣＣＴＴＧＧＡＡＡＧＧＡＡＣＡＡＧＡＣＣＣＡＡＣＡＧＧＧＧＴＡＧＡＡＣＣＴＧＧＡＧＡＴＣＡＴＣＴＧＡＡＡＧＡＧＡＧＡＴＣＡＡＴＧＣＴＣＡＧＴＴＡＴＧＧＡＡＡＴＧＴＴＣＴＴＧＡＣＴＴＡＡＡＣＣＡＣＣＴＧＧＡＴＡＴＴＧＡＴＧＡＧＣＣＡＡＣＡＧＧＡＣＡＧＡＣＡＧＣＡＧＡＣＴＧＧＣＴＧＧＧＣＡＴＴＧＴＴＡＴＣＴＡＴＣＴＣＡＣＡＴＣＣＴＴＴＧＴＴＧＴＣＣＣＧＡＴＡＣＴＴＣＴＧＡＡＡＧＣＣＣＴＧＴＡＣＡＴＧＴＴＡＡＣＡＡＣＡＡＧＡＧＧＧＡＧＧＣＡＧＡＣＣＡＣＣＡＡＧＧＡＣＡＡＴＡＡＡＧＧＡＡＣＴＣＧＧＡＴＴＣＧＡＴＴＣＡＡＧＧＡＴＧＡＴＡＧＣＴＣＣＴＴＣＧＡＧＧＡＴＧＴＣＡＡＴＧＧＣＡＴＴＣＧＧＡＡＧＣＣＧＡＡＡＣＡＴＣＴＡＴＡＴＧＴＧＴＣＣＴＴＡＣＣＡＡＡＴＧＣＡＣＡＧＴＣＡＡＧＴＡＴＧＡＡＡＧＣＡＧＡＡＧＡＧＡＴＴＡＣＡＣＣＴＧＧＴＡＧＡＴＡＴＡＧＡＡＣＡＧＣＡＡＴＴＴＧＴＧＧＡＣＴＴＴＡＣＣＣＴＧＣＡＣＡＡＡＴＴＡＡＧＧＣＡＡＧＡＣＡＧＡＴＧＡＴＴＡＧＴＣＣＡＧＴＣＡＴＧＡＧＴＧＴＡＡＴＣＧＧＡＴＴＣＴＴＧＧＣＴＴＴＧＧＣＡＡＡＡＧＡＴＴＧＧＡＧＴＧＡＣＣＧＣＡＴＴＧＡＧＣＡＧＴＧＧＴＴＡＡＧＴＧＡＡＣＣＧＴＧＴＡＡＧＣＴＴＣＴＴＣＣＡＧＡＴＡＣＡＧＣＡＧＣＡＧＴＴＡＧＣＣＴＴＣＴＴＧＧＴＧＧＴＣＣＴＧＣＡＡＣＣＡＡＣＡＧＧＧＡＣＴＡＴＴＴＡＣＧＧＣＡＧＣＧＡＣＡＡＧＴＡＧＣＡＴＴＧＧＧＣＡＡＣＡＴＧＧＡＡＡＣＡＡＡＡＧＡＧＴＣＴＡＡＧＧＣＴＡＴＡＣＧＣＣＡＡＣＡＴＧＣＡＧＡＡＧＣＡＧＣＡＧＧＣＴＧＴＡＧＴＡＴＧＡＴＴＧＡＧＧＡＣＡＴＴＧＡＧＴＣＡＣＣＡＴＣＡＴＣＡＡＴＡＴＧＧＧＴＧＴＴＴＧＣＴＧＧＧＧＣＡＣＣＧＧＡＣＣＧＣＴＧＴＣＣＡＣＣＡＡＣＡＴＧＴＣＴＣＴＴＴＡＴＴＧＣＡＧＧＴＡＴＧＧＣＴＧＡＧＣＴＴＧＧＧＧＣＡＴＴＴＴＴＴＴＣＣＡ ＴＣＣＴＧＣＡＧＧＡＣＡＴＧＣＧＡＡＡＴＡＣＡＡＴＴＡＴＧＧＣＡＴＣＣＡＡＧＡＣＡＧＴＴＧＧＡＡＣＣＴＣＴＧＡＧＧＡＧＡＡＧＣＴＡＣＧＧＡＡＧＡＡＡＴＣＣＴＣＡＴＴＣＴＡＴＣＡＧＴＣＴＴＡＴＣＴＣＡＧＧＡＧＡＡＣＡＣＡＡＴＣＡＡＴＧＧＧＡＡＴＡＣＡＡＣＴＧＧＡＴＣＡＧＡＧＧＡＴＡＡＴＴＧＴＧＣＴＣＴＴＣＡＴＧＧＴＡＧＣＣＴＧＧＧＧＧＡＡＡＧＡＡＧＣＡＧＴＧＧＡＴＡＡＣＴＴＣＣＡＣＣＴＡＧＧＡＧＡＴＧＡＴＡＴＧＧＡＣＣＣＴＧＡＧＣＴＧＣＧＡＡＣＡＣＴＡＧＣＡＣＡＧＡＧＣＣＴＧＡＴＴＧＡＴＧＴＴＡＡＡＧＴＧＡＡＧＧＡＡＡＴＴＴＣＣＡＡＣＣＡＡＧＡＧＣＣＴＴＴＡＡＡＡＣＴＡ（配列番号６）。

Translation of the nucleic acid sequence of SEQ ID NO:6 yields the hantavirus NP polypeptide sequence, which is represented by (SEQ ID NO:7):
ＭＡＴＭＥＥＬＱＲＥＩＮＡＨＥＧＱＬＶＩＡＲＱＫＶＲＤＡＥＫＱＹＥＫＤＰＤＥＬＮＫＲＡＬＴＤＲＥＧＶＡＶＳＩＱＡＫＩＤＥＬＫＲＱＬＡＤＲＩＡＴＧＫＮＬＧＫＥＱＤＰＴＧＶＥＰＧＤＨＬＫＥＲＳＭＬＳＹＧＮＶＬＤＬＮＨＬＤＩＤＥＰＴＧＱＴＡＤＷＬＧＩＶＩＹＬＴＳＦＶＶＰＩＬＬＫＡＬＹＭＬＴＴＲＧＲＱＴＴＫＤＮＫＧＴＲＩＲＦＫＤＤＳＳＦＥＤＶＮＧＩＲＫＰＫＨＬＹＶＳＬＰＮＡＱＳＳＭＫＡＥＥＩＴＰＧＲＹＲＴＡＩＣＧＬＹＰＡＱＩＫＡＲＱＭＩＳＰＶＭＳＶＩＧＦＬＡＬＡＫＤＷＳＤＲＩＥＱＷＬＳＥＰＣＫＬＬＰＤＴＡＡＶＳＬＬＧＧＰＡＴＮＲＤＹＬＲＱＲＱＶＡＬＧＮＭＥＴＫＥＳＫＡＩＲＱＨＡＥＡＡＧＣＳＭＩＥＤＩＥＳＰＳＳＩＷＶＦＡＧＡＰＤＲＣＰＰＴＣＬＦＩＡＧＭＡＥＬＧＡＦＦＳＩＬＱＤＭＲＮＴＩＭＡＳＫＴＶＧＴＳＥＥＫＬＲＫＫＳＳＦＹＱＳＹＬＲＲＴＱＳＭＧＩＱＬＤＱＲＩＩＶＬＦＭＶＡＷＧＫＥＡＶＤＮＦＨＬＧＤＤＭＤＰＥＬＲＴＬＡＱＳＬＩＤＶＫＶＫＥＩＳＮＱＥＰＬＫＬ（配列番号７）。

A reference nucleic acid sequence for hantavirus NP can be provided by SEQ ID NO:8, which corresponds to nucleic acid residues 319-1323 of SEQ ID NO:5.
ＡＴＧＣＴＣＡＧＴＴＡＴＧＧＡＡＡＴＧＴＴＣＴＴＧＡＣＴＴＡＡＡＣＣＡＣＣＴＧＧＡＴＡＴＴＧＡＴＧＡＧＣＣＡＡＣＡＧＧＡＣＡＧＡＣＡＧＣＡＧＡＣＴＧＧＣＴＧＧＧＣＡＴＴＧＴＴＡＴＣＴＡＴＣＴＣＡＣＡＴＣＣＴＴＴＧＴＴＧＴＣＣＣＧＡＴＡＣＴＴＣＴＧＡＡＡＧＣＣＣＴＧＴＡＣＡＴＧＴＴＡＡＣＡＡＣＡＡＧＡＧＧＧＡＧＧＣＡＧＡＣＣＡＣＣＡＡＧＧＡＣＡＡＴＡＡＡＧＧＡＡＣＴＣＧＧＡＴＴＣＧＡＴＴＣＡＡＧＧＡＴＧＡＴＡＧＣＴＣＣＴＴＣＧＡＧＧＡＴＧＴＣＡＡＴＧＧＣＡＴＴＣＧＧＡＡＧＣＣＧＡＡＡＣＡＴＣＴＡＴＡＴＧＴＧＴＣＣＴＴＡＣＣＡＡＡＴＧＣＡＣＡＧＴＣＡＡＧＴＡＴＧＡＡＡＧＣＡＧＡＡＧＡＧＡＴＴＡＣＡＣＣＴＧＧＴＡＧＡＴＡＴＡＧＡＡＣＡＧＣＡＡＴＴＴＧＴＧＧＡＣＴＴＴＡＣＣＣＴＧＣＡＣＡＡＡＴＴＡＡＧＧＣＡＡＧＡＣＡＧＡＴＧＡＴＴＡＧＴＣＣＡＧＴＣＡＴＧＡＧＴＧＴＡＡＴＣＧＧＡＴＴＣＴＴＧＧＣＴＴＴＧＧＣＡＡＡＡＧＡＴＴＧＧＡＧＴＧＡＣＣＧＣＡＴＴＧＡＧＣＡＧＴＧＧＴＴＡＡＧＴＧＡＡＣＣＧＴＧＴＡＡＧＣＴＴＣＴＴＣＣＡＧＡＴＡＣＡＧＣＡＧＣＡＧＴＴＡＧＣＣＴＴＣＴＴＧＧＴＧＧＴＣＣＴＧＣＡＡＣＣＡＡＣＡＧＧＧＡＣＴＡＴＴＴＡＣＧＧＣＡＧＣＧＡＣＡＡＧＴＡＧＣＡＴＴＧＧＧＣＡＡＣＡＴＧＧＡＡＡＣＡＡＡＡＧＡＧＴＣＴＡＡＧＧＣＴＡＴＡＣＧＣＣＡＡＣＡＴＧＣＡＧＡＡＧＣＡＧＣＡＧＧＣＴＧＴＡＧＴＡＴＧＡＴＴＧＡＧＧＡＣＡＴＴＧＡＧＴＣＡＣＣＡＴＣＡＴＣＡＡＴＡＴＧＧＧＴＧＴＴＴＧＣＴＧＧＧＧＣＡＣＣＧＧＡＣＣＧＣＴＧＴＣＣＡＣＣＡＡＣＡＴＧＴＣＴＣＴＴＴＡＴＴＧＣＡＧＧＴＡＴＧＧＣＴＧＡＧＣＴＴＧＧＧＧＣＡＴＴＴＴＴＴＴＣＣＡＴＣＣＴＧＣＡＧＧＡＣＡＴＧＣＧＡＡＡＴＡＣＡＡＴＴＡＴＧＧＣＡＴＣＣＡＡＧＡＣＡＧＴＴＧＧＡＡＣＣＴＣＴＧＡＧＧＡＧＡＡＧＣＴＡＣＧＧＡＡＧＡＡＡＴＣＣＴＣＡＴＴＣＴＡＴＣＡＧＴＣＴＴＡＴＣＴＣＡＧＧＡＧＡＡＣＡＣＡＡＴＣＡＡＴＧＧＧＡＡＴＡＣＡＡＣＴＧＧＡＴＣＡＧＡＧＧＡＴＡＡＴＴＧＴＧＣＴＣＴＴＣＡＴＧＧＴＡＧＣＣＴＧＧＧＧＧＡＡＡＧＡＡＧＣＡＧＴＧＧＡＴＡＡＣＴＴＣＣＡＣＣＴＡＧＧＡＧＡＴＧＡＴＡＴＧＧＡＣＣＣＴＧＡＧＣＴＧＣＧＡＡＣＡＣＴＡＧＣＡＣＡＧＡＧＣＣＴＧＡＴＴＧＡＴＧＴＴＡＡＡＧＴＧＡＡＧＧＡＡＡＴＴＴＣＣＡＡＣＣＡＡＧＡＧＣＣＴＴＴＡＡ AACTA (SEQ ID NO: 8).

The inventors have generated a nucleic acid sequence encoding hantavirus NP that has been optimized for expression in Homo sapiens (see SEQ ID NO: 9):
ＡＴＧＣＴＧＡＧＣＴＡＣＧＧＣＡＡＣＧＴＧＣＴＧＧＡＴＣＴＧＡＡＣＣＡＣＣＴＧＧＡＴＡＴＣＧＡＣＧＡＧＣＣＡＡＣＡＧＧＡＣＡＧＡＣＣＧＣＴＧＡＴＴＧＧＣＴＧＧＧＣＡＴＣＧＴＧＡＴＣＴＡＣＣＴＧＡＣＣＴＣＣＴＴＴＧＴＧＧＴＧＣＣＴＡＴＴＣＴＧＣＴＣＡＡＡＧＣＣＣＴＣＴＡＴＡＴＧＣＴＧＡＣＡＡＣＡＣＧＣＧＧＡＡＧＧＣＡＧＡＣＣＡＣＣＡＡＡＧＡＴＡＡＣＡＡＡＧＧＣＡＣＣＣＧＧＡＴＣＡＧＧＴＴＴＡＡＧＧＡＣＧＡＣＡＧＣＴＣＣＴＴＴＧＡＧＧＡＴＧＴＣＡＡＣＧＧＣＡＴＣＣＧＧＡＡＡＣＣＴＡＡＧＣＡＣＣＴＣＴＡＴＧＴＧＴＣＴＣＴＧＣＣＣＡＡＴＧＣＡＣＡＧＴＣＣＴＣＣＡＴＧＡＡＧＧＣＡＧＡＡＧＡＧＡＴＣＡＣＡＣＣＡＧＧＣＣＧＧＴＡＣＡＧＡＡＣＣＧＣＣＡＴＣＴＧＴＧＧＡＣＴＧＴＡＴＣＣＴＧＣＡＣＡＡＡＴＣＡＡＡＧＣＣＣＧＧＣＡＧＡＴＧＡＴＣＡＧＣＣＣＣＧＴＧＡＴＧＴＣＣＧＴＴＡＴＣＧＧＡＴＴＣＣＴＧＧＣＴＣＴＧＧＣＣＡＡＡＧＡＴＴＧＧＡＧＣＧＡＣＡＧＧＡＴＣＧＡＧＣＡＧＴＧＧＣＴＧＡＧＣＧＡＧＣＣＴＴＧＣＡＡＧＣＴＧＣＴＴＣＣＴＧＡＴＡＣＡＧＣＣＧＣＴＧＴＧＴＣＡＣＴＧＣＴＴＧＧＣＧＧＣＣＣＴＧＣＣＡＣＡＡＡＣＡＧＡＧＡＴＴＡＣＣＴＧＡＧＡＣＡＧＡＧＡＣＡＧＧＴＧＧＣＡＣＴＧＧＧＣＡＡＣＡＴＧＧＡＡＡＣＡＡＡＡＧＡＧＡＧＣＡＡＧＧＣＣＡＴＣＣＧＧＣＡＧＣＡＴＧＣＣＧＡＡＧＣＴＧＣＴＧＧＣＴＧＴＡＧＣＡＴＧＡＴＣＧＡＧＧＡＴＡＴＣＧＡＧＴＣＣＣＣＴＡＧＣＴＣＣＡＴＴＴＧＧＧＴＧＴＴＣＧＣＡＧＧＧＧＣＣＣＣＡＧＡＴＡＧＡＴＧＴＣＣＡＣＣＡＡＣＡＴＧＣＣＴＧＴＴＣＡＴＴＧＣＣＧＧＣＡＴＧＧＣＴＧＡＡＣＴＧＧＧＡＧＣＴＴＴＴＴＴＣＡＧＣＡＴＣＣＴＣＣＡＧＧＡＴＡＴＧＣＧＣＡＡＣＡＣＧＡＴＴＡＴＧＧＣＣＴＣＣＡＡＧＡＣＡＧＴＧＧＧＡＡＣＣＡＧＣＧＡＧＧＡＡＡＡＧＣＴＧＣＧＧＡＡＧＡＡＡＡＧＣＡＧＣＴＴＴＴＡＣＣＡＧＴＣＴＴＡＣＣＴＧＡＧＧＣＧＧＡＣＣＣＡＧＴＣＣＡＴＧＧＧＧＡＴＣＣＡＡＣＴＧＧＡＴＣＡＧＣＧＧＡＴＣＡＴＴＧＴＧＣＴＧＴＴＴＡＴＧＧＴＣＧＣＴＴＧＧＧＧＡＡＡＡＧＡＧＧＣＴＧＴＣＧＡＴＡＡＣＴＴＣＣＡＣＣＴＧＧＧＡＧＡＴＧＡＴＡＴＧＧＡＴＣＣＴＧＡＡＣＴＧＣＧＧＡＣＣＣＴＧＧＣＴＣＡＧＴＣＣＣＴＧＡＴＣＧＡＴＧＴＧＡＡＡＧＴＧＡＡＡＧＡＡＡＴＴＡＧＴＡＡＴＣＡＡＧＡＡＣＣＣＣＴＣＡ AGCTG (SEQ ID NO: 9).

Nucleic acid sequences comprising SEQ ID NO: 8 or 9 are particularly well suited for use in vectors of the invention that also encode nucleoproteins from Hantavirus strains other than Hantavirus, such as Seoul virus. We have determined that the 94 N-terminal amino acids of the wild-type htan virus nucleoprotein show high sequence similarity to the N-terminus of the wild-type nucleoprotein from Soul virus, and sequence differences are present in this region. We have determined that both sequences contain closely related amino acids. Residue 95 of the wild-type Hantan virus nucleoprotein sequence was identified as the first residue significantly different from the corresponding residue of the wild-type nucleoprotein sequence from Seoul virus. The present inventors have determined that the nucleic acid encoding the 94 N-terminal amino acids of the wild-type nucleoprotein from Hantan virus is the nucleoprotein from Seoul virus (or at least the 94 N-terminal amino acids of the wild-type nucleoprotein from Seoul virus). amino acids, or antigenic fragments thereof) are also considered to be substantially antigenically redundant. Therefore, the present inventors proposed that the nucleic acid encoding the 94 N-terminal amino acids of the wild-type Hantan virus nucleoprotein be transformed into a Seoul virus-derived nucleoprotein (or Seoul virus-derived nucleoprotein) without sacrificing antigenic diversity. It is contemplated that at least 94 N-terminal amino acids of the wild-type nucleoprotein, or an antigenic fragment thereof) can also be omitted from the encoding vector. Removal of unnecessary nucleic acid sequences is generally advantageous in the design of vector constructs (eg, MVA constructs) as it can enhance vector stability.

For the above reasons, we have identified the nucleic acid encoding the 94 N-terminal amino acids of the Seoul virus nucleoprotein, in particular the Hantan virus nucleoprotein (or at least the 94 amino acids of the wild-type nucleoprotein from Hantan virus). (or an antigenic fragment thereof) can be omitted from the encoding vector to achieve similar advantages.

Translation of the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:9 yields the hantavirus NP polypeptide sequence, which is represented by (SEQ ID NO:10):
ＭＬＳＹＧＮＶＬＤＬＮＨＬＤＩＤＥＰＴＧＱＴＡＤＷＬＧＩＶＩＹＬＴＳＦＶＶＰＩＬＬＫＡＬＹＭＬＴＴＲＧＲＱＴＴＫＤＮＫＧＴＲＩＲＦＫＤＤＳＳＦＥＤＶＮＧＩＲＫＰＫＨＬＹＶＳＬＰＮＡＱＳＳＭＫＡＥＥＩＴＰＧＲＹＲＴＡＩＣＧＬＹＰＡＱＩＫＡＲＱＭＩＳＰＶＭＳＶＩＧＦＬＡＬＡＫＤＷＳＤＲＩＥＱＷＬＳＥＰＣＫＬＬＰＤＴＡＡＶＳＬＬＧＧＰＡＴＮＲＤＹＬＲＱＲＱＶＡＬＧＮＭＥＴＫＥＳＫＡＩＲＱＨＡＥＡＡＧＣＳＭＩＥＤＩＥＳＰＳＳＩＷＶＦＡＧＡＰＤＲＣＰＰＴＣＬＦＩＡＧＭＡＥＬＧＡＦＦＳＩＬＱＤＭＲＮＴＩＭＡＳＫＴＶＧＴＳＥＥＫＬＲＫＫＳＳＦＹＱＳＹＬＲＲＴＱＳＭＧＩＱＬＤＱＲＩＩＶＬＦＭＶＡＷＧＫＥＡＶＤＮＦＨＬＧＤＤＭＤＰＥＬＲＴＬＡＱＳＬＩＤＶＫＶＫＥＩＳＮＱＥＰＬＫＬ（配列番号１０）。

As used herein, the term "antigenic fragment" means a peptide or protein fragment of hantavirus NP that retains the ability to induce an immune response in an individual compared to the reference hantavirus NP. An antigenic fragment may therefore comprise at least one epitope of the reference protein. By way of example, antigenic fragments of the invention have at least 5, 10, 15, 20, 25, 30, 35, 5, 10, 15, 20, 25, 30, 35, 5, 10, 15, 20, 25, 30, 35, 35, 35, 35, 35, 34, 35, 35, 35, 35, 35, 35, 35, 35, 35, 35, 35, 4, 4, 4, 4, 4, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, respectively, It may comprise (or consist of) a peptide sequence having 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300 amino acids. An antigenic fragment is at least 10 contiguous amino acid residues from the sequence of a reference protein (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 , 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275 or 300 contiguous amino acid residues).

An antigenic fragment of a reference protein can have common antigenic cross-reactivity and/or substantially the same in vivo biological activity as the reference protein. For example, an antibody that can bind an antigenic fragment of a reference protein can also bind the reference protein itself. As a further example, reference proteins and antigenic fragments thereof may be used to target T lymphocytes (e.g., CD4+, CD8+, effector T cells or memory T cells, e.g., TEM or TCM) previously exposed to antigenic components of hantavirus infection. ) may share a common ability to induce a “recall response”.

In one aspect, the invention provides a viral or bacterial vector comprising a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof and capable of inducing an immune response in a subject.

In one embodiment, the nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof is at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity.

In one embodiment, the nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof is at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90) the nucleic acid sequence of SEQ ID NO:3. , 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof is at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity.

In one embodiment, the nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof is at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90) the nucleic acid sequence of SEQ ID NO:9. , 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity.

'Peptide pool 4' induced very strong antigen-specific T cell responses (see Examples). The amino acid sequence represented by peptide pool 4 corresponds to SEQ ID NO:11.
LYPAQIKARNMVSPVMSVVGFLALAKDWTSRIEEWLGAPCKFMAESPIAGSLSGNPVNRDYIRQRQGALAGMEPKEFQA (SEQ ID NO: 11).

The amino acid sequence of SEQ ID NO:11 is encoded by nucleic acid residues 655-891 of SEQ ID NO:1 (see SEQ ID NO:15) and by residues 613-849 of SEQ ID NO:2 (see SEQ ID NO:16). , and residues 613-849 of SEQ ID NO:3 (see SEQ ID NO:17).
ＣＴＡＴＡＣＣＣＴＧＣＡＣＡＧＡＴＡＡＡＧＧＣＡＡＧＧＡＡＣＡＴＧＧＴＡＡＧＣＣＣＴＧＴＣＡＴＧＡＧＴＧＴＡＧＴＴＧＧＧＴＴＴＴＴＧＧＣＡＣＴＧＧＣＡＡＡＡＧＡＣＴＧＧＡＣＡＴＣＴＡＧＡＡＴＴＧＡＡＧＡＡＴＧＧＣＴＴＧＧＴＧＣＡＣＣＣＴＧＣＡＡＧＴＴＣＡＴＧＧＣＡＧＡＧＴＣＴＣＣＣＡＴＴＧＣＣＧＧＧＡＧＣＴＴＡＴＣＴＧＧＧＡＡＴＣＣＴＧＴＧＡＡＴＣＧＴＧＡＴＴＡＴＡＴＣＡＧＡＣＡＧＡＧＡＣＡＡＧＧＴＧＣＡＣＴＴＧＣＡＧＧＧＡＴＧＧＡＧＣＣＡＡＡＡＧＡＡＴＴＴＣＡＡＧＣＴ（配列番号１５）
ＣＴＡＴＡＣＣＣＴＧＣＡＣＡＧＡＴＡＡＡＧＧＣＡＡＧＧＡＡＣＡＴＧＧＴＡＡＧＣＣＣＴＧＴＣＡＴＧＡＧＴＧＴＡＧＴＴＧＧＧＴＴＴＴＴＧＧＣＡＣＴＧＧＣＡＡＡＡＧＡＣＴＧＧＡＣＡＴＣＴＡＧＡＡＴＴＧＡＡＧＡＡＴＧＧＣＴＴＧＧＴＧＣＡＣＣＣＴＧＣＡＡＧＴＴＣＡＴＧＧＣＡＧＡＧＴＣＴＣＣＣＡＴＴＧＣＣＧＧＧＡＧＣＴＴＡＴＣＴＧＧＧＡＡＴＣＣＴＧＴＧＡＡＴＣＧＴＧＡＴＴＡＴＡＴＣＡＧＡＣＡＧＡＧＡＣＡＡＧＧＴＧＣＡＣＴＴＧＣＡＧＧＧＡＴＧＧＡＧＣＣＡＡＡＡＧＡＡＴＴＴＣＡＡＧＣＴ（配列番号１６）
ＣＴＧＴＡＣＣＣＣＧＣＴＣＡＧＡＴＣＡＡＧＧＣＣＡＧＡＡＡＣＡＴＧＧＴＧＴＣＣＣＣＡＧＴＧＡＴＧＡＧＣＧＴＣＧＴＧＧＧＡＴＴＴＣＴＧＧＣＣＣＴＧＧＣＴＡＡＧＧＡＣＴＧＧＡＣＣＡＧＣＡＧＧＡＴＴＧＡＧＧＡＡＴＧＧＣＴＧＧＧＡＧＣＣＣＣＴＴＧＣＡＡＧＴＴＴＡＴＧＧＣＣＧＡＧＴＣＴＣＣＴＡＴＣＧＣＣＧＧＣＡＧＣＣＴＧＴＣＴＧＧＣＡＡＣＣＣＣＧＴＧＡＡＴＡＧＡＧＡＣＴＡＣＡＴＣＡＧＡＣＡＧＡＧＧＣＡＧＧＧＣＧＣＴＣＴＧＧＣＣＧＧＡＡＴＧＧＡＡＣＣＣＡＡＡＧＡＡＴＴＴＣＡＧＧＣＣ（配列番号１７）

In one embodiment, the nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof is at least 70% (e.g., at least 70, 75, 80, 82, 84, 86) the nucleic acid sequence of SEQ ID NO: 15, 16 or 17. , 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof is at least 10 contiguous nucleic acid residues from the sequence of SEQ ID NO: 15, 16 or 17 (e.g., SEQ ID NO: 15, 16 or at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65 nucleic acids of 17).

'Peptide Pool 9' also induced very strong antigen-specific T cell responses. The amino acid sequence represented by peptide pool 9 corresponds to SEQ ID NO:12.
IKARQMISPVMSVIGFLALAKDWSDRIEQWLSEPCKLLPDTAAVSLLLGGPATNRDYLRQRQVALGNMETKESKAIRQHA (SEQ ID NO: 12)

The amino acid sequence of SEQ ID NO:12 is encoded by nucleic acid residues 664-900 of SEQ ID NO:5 (see SEQ ID NO:18) and by residues 628-864 of SEQ ID NO:6 (see SEQ ID NO:19). , residues 346-582 of SEQ ID NO:8 (see SEQ ID NO:20) and by residues 346-582 of SEQ ID NO:9 (see SEQ ID NO:21).
ＡＴＴＡＡＧＧＣＡＡＧＡＣＡＧＡＴＧＡＴＴＡＧＴＣＣＡＧＴＣＡＴＧＡＧＴＧＴＡＡＴＣＧＧＡＴＴＣＴＴＧＧＣＴＴＴＧＧＣＡＡＡＡＧＡＴＴＧＧＡＧＴＧＡＣＣＧＣＡＴＴＧＡＧＣＡＧＴＧＧＴＴＡＡＧＴＧＡＡＣＣＧＴＧＴＡＡＧＣＴＴＣＴＴＣＣＡＧＡＴＡＣＡＧＣＡＧＣＡＧＴＴＡＧＣＣＴＴＣＴＴＧＧＴＧＧＴＣＣＴＧＣＡＡＣＣＡＡＣＡＧＧＧＡＣＴＡＴＴＴＡＣＧＧＣＡＧＣＧＡＣＡＡＧＴＡＧＣＡＴＴＧＧＧＣＡＡＣＡＴＧＧＡＡＡＣＡＡＡＡＧＡＧＴＣＴＡＡＧＧＣＴＡＴＡＣＧＣＣＡＡＣＡＴＧＣＡ（配列番号１８）
ＡＴＴＡＡＧＧＣＡＡＧＡＣＡＧＡＴＧＡＴＴＡＧＴＣＣＡＧＴＣＡＴＧＡＧＴＧＴＡＡＴＣＧＧＡＴＴＣＴＴＧＧＣＴＴＴＧＧＣＡＡＡＡＧＡＴＴＧＧＡＧＴＧＡＣＣＧＣＡＴＴＧＡＧＣＡＧＴＧＧＴＴＡＡＧＴＧＡＡＣＣＧＴＧＴＡＡＧＣＴＴＣＴＴＣＣＡＧＡＴＡＣＡＧＣＡＧＣＡＧＴＴＡＧＣＣＴＴＣＴＴＧＧＴＧＧＴＣＣＴＧＣＡＡＣＣＡＡＣＡＧＧＧＡＣＴＡＴＴＴＡＣＧＧＣＡＧＣＧＡＣＡＡＧＴＡＧＣＡＴＴＧＧＧＣＡＡＣＡＴＧＧＡＡＡＣＡＡＡＡＧＡＧＴＣＴＡＡＧＧＣＴＡＴＡＣＧＣＣＡＡＣＡＴＧＣＡ（配列番号１９）
ＡＴＴＡＡＧＧＣＡＡＧＡＣＡＧＡＴＧＡＴＴＡＧＴＣＣＡＧＴＣＡＴＧＡＧＴＧＴＡＡＴＣＧＧＡＴＴＣＴＴＧＧＣＴＴＴＧＧＣＡＡＡＡＧＡＴＴＧＧＡＧＴＧＡＣＣＧＣＡＴＴＧＡＧＣＡＧＴＧＧＴＴＡＡＧＴＧＡＡＣＣＧＴＧＴＡＡＧＣＴＴＣＴＴＣＣＡＧＡＴＡＣＡＧＣＡＧＣＡＧＴＴＡＧＣＣＴＴＣＴＴＧＧＴＧＧＴＣＣＴＧＣＡＡＣＣＡＡＣＡＧＧＧＡＣＴＡＴＴＴＡＣＧＧＣＡＧＣＧＡＣＡＡＧＴＡＧＣＡＴＴＧＧＧＣＡＡＣＡＴＧＧＡＡＡＣＡＡＡＡＧＡＧＴＣＴＡＡＧＧＣＴＡＴＡＣＧＣＣＡＡＣＡＴＧＣＡ（配列番号２０）
ＡＴＣＡＡＡＧＣＣＣＧＧＣＡＧＡＴＧＡＴＣＡＧＣＣＣＣＧＴＧＡＴＧＴＣＣＧＴＴＡＴＣＧＧＡＴＴＣＣＴＧＧＣＴＣＴＧＧＣＣＡＡＡＧＡＴＴＧＧＡＧＣＧＡＣＡＧＧＡＴＣＧＡＧＣＡＧＴＧＧＣＴＧＡＧＣＧＡＧＣＣＴＴＧＣＡＡＧＣＴＧＣＴＴＣＣＴＧＡＴＡＣＡＧＣＣＧＣＴＧＴＧＴＣＡＣＴＧＣＴＴＧＧＣＧＧＣＣＣＴＧＣＣＡＣＡＡＡＣＡＧＡＧＡＴＴＡＣＣＴＧＡＧＡＣＡＧＡＧＡＣＡＧＧＴＧＧＣＡＣＴＧＧＧＣＡＡＣＡＴＧＧＡＡＡＣＡＡＡＡＧＡＧＡＧＣＡＡＧＧＣＣＡＴＣＣＧＧＣＡＧＣＡＴＧＣＣ（配列番号２１）

In one embodiment, the nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof is at least 70% (e.g., at least 70, 75, 80, 82, 84) the nucleic acid sequence of SEQ ID NO: 18, 19, 20 or 21. , 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof is at least 10 contiguous nucleic acid residues from the sequence of SEQ ID NO: 18, 19, 20 or 21 (e.g., SEQ ID NO: 18, 19, 20 or 21 at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65 nucleic acids).

As demonstrated herein, peptide pools 4 and 9 induced very strong antigen-specific T cell responses. By aligning the polypeptide sequences represented by "Peptide Pool 4" and the polypeptide sequences represented by "Peptide Pool 9", we obtained the Regions of high sequence identity were identified. Without wishing to be bound by theory, the inventors believe that the amino acid sequences of SEQ ID NOs: 13 and 14 are effective in eliciting particularly strong antigen-specific T cell responses observed with peptide pools 4 and 9, respectively. I believe it will play an important role.
SPVMSVVGFLALAKD (SEQ ID NO: 13)
PVMSVIGFLALAKDW (SEQ ID NO: 14)

SEQ ID NO: 13 is specifically encoded by nucleic acid residues 691-735 of SEQ ID NO: 1 (see SEQ ID NO: 22), by residues 649-693 of SEQ ID NO: 2 (see SEQ ID NO: 23), and Encoded by residues 649-693 of SEQ ID NO:3 (see SEQ ID NO:24).
AGCCCTGTCATGAGTGTAGTTGGGTTTTTGGCACTGGCAAAAGAC (SEQ ID NO: 22)
AGCCCTGTCATGAGTGTAGTTGGGTTTTTGGCACTGGCAAAGAC (SEQ ID NO: 23)
TCCCCAGTGATGAGCGTCGTGGGATTTCTGGCCCTGGCTAAGGAC (SEQ ID NO: 24)

In one embodiment, the nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof is at least 70% (e.g., at least 70, 75, 80, 82, 84, 86) the nucleic acid sequence of SEQ ID NO: 22, 23 or 24. , 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity.

SEQ ID NO: 14 is specifically encoded by nucleic acid residues 688-732 of SEQ ID NO: 5 (see SEQ ID NO: 25) and encoded by residues 652-696 of SEQ ID NO: 6 (see SEQ ID NO: 26). Encoded by residues 370-414 of number 8 (see SEQ ID NO:27) and by residues 370-414 of SEQ ID NO:9 (see SEQ ID NO:28).
CCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGG (SEQ ID NO: 25)
CCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGG (SEQ ID NO: 26)
CCAGTCATGAGTGTAATCGGATTCTTGGCTTTGGCAAAAGATTGG (SEQ ID NO: 27)
CCCGTGATGTCCGTTATCGGATTCCTGGCTCTGGCCCAAAGATTGG (SEQ ID NO: 28)

In one embodiment, the nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof is at least 70% (e.g., at least 70, 75, 80, 82, 84) the nucleic acid sequence of SEQ ID NO: 25, 26, 27 or 28. , 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence,
(A) the first nucleic acid sequence is at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, (B) said second nucleic acid sequence has at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence,
(A) the first nucleic acid sequence is at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity; and (B) said second nucleic acid sequence is SEQ ID NO: 18, 19, 20, 21, 25 , 26, 27 or 28 at least 70% (e.g. at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity is provided by a nucleic acid sequence having a

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence,
(A) said first nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 15, 16, 17, 22, 23 or 24; and (B) said second The nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 18, 19, 20, 21, 25, 26, 27 or 28.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence,
(A) said first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:24; and (B) said second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:28. % sequence identity.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence,
(A) said first nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 22 or 23; and (B) said second nucleic acid sequence is SEQ ID NO: 25, A nucleic acid sequence having at least 70% sequence identity with a nucleic acid sequence selected from 26 or 27.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence,
(A) said first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 17; and (B) said second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 21. % sequence identity.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence,
(A) said first nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 15 or 16; and (B) said second nucleic acid sequence is SEQ ID NO: 18, have at least 70% sequence identity with a nucleic acid sequence selected from 19 or 20;

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence,
(A) said first nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 1, 2 or 3; and (B) said second nucleic acid sequence has SEQ ID NO: Has at least 70% sequence identity with a nucleic acid sequence selected from 5, 6, 8 or 9.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence,
(A) said first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:3; and (B) said second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:9. % sequence identity.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence,
(A) said first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:2; and (B) said second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:8. % sequence identity.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence,
(A) said first nucleic acid has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:2; and (B) said second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:6. sequence identity.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a first nucleic acid sequence and a second nucleic acid sequence,
(A) said first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:1; and (B) said second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:5. % sequence identity.

In one embodiment, the first nucleic acid sequence is located 5' to the second nucleic acid sequence. In one embodiment, the second nucleic acid sequence is located 5' to the first nucleic acid sequence.

In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof is SEQ ID NO: 29 and at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity.
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In one embodiment, the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof is SEQ ID NO: 30 and at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity.
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The inventors have found that hantavirus NP encoded by the nucleic acid sequences of the invention can be used to generate an effective immune response against hantavirus in an individual. In particular, we have found that delivery of hantavirus NP to subjects using bacterial or viral vectors, such as non-replicating poxvirus vectors or adenoviral vectors, results in highly effective immune responses against hantavirus. I found out.

A vector is a tool that can be used as a vector for the delivery of genetic material to target cells. By way of example, viral vectors function as antigen delivery vehicles and also have the ability to activate the innate immune system by binding to cell surface molecules that recognize viral elements. Recombinant viral vectors carrying nucleic acids encoding a given antigen can be prepared. A viral vector can then be used to deliver the nucleic acid to a target cell where the encoded antigen is produced and then presented to the immune system by the target cell's own molecular machinery. As "non-self," the antigens produced generate an adaptive immune response in the target subject. Advantageously, the vectors of the invention are demonstrated herein to provide a protective immune response.

Viral vectors suitable for use in the present invention include poxvirus vectors (such as non-replicating poxvirus vectors), adenovirus vectors, and influenza virus vectors.

In certain embodiments, a "viral vector" can be a virus-like particle (VLP). VLPs are lipid enveloped particles containing viral proteins. Certain viral proteins have the inherent ability to self-assemble, a process in which they bud from the cell membrane as independent membrane-enveloped particles. VLPs are easy to purify and can be used, for example, to present viral antigens. VLPs are therefore suitable for use in immunogenic compositions such as those described below. In certain embodiments, a "viral vector" is not a virus-like particle.

Bacterial vectors can also be used as antigen delivery vehicles. A recombinant bacterial vector can be prepared carrying a nucleic acid encoding a given antigen. A recombinant bacterial vector can express an antigen on its surface. After administration to a subject, the bacterial vector colonizes antigen-presenting cells (eg, dendritic cells or macrophages). An antigen-specific immune response is induced. The immune response can be a cellular (T-cell) immune response or can include both humoral (eg, B-cell) and cellular (T-cell) immune responses. Examples of bacteria suitable for use as recombinant bacterial vectors include E. coli, Shigella, Salmonella (eg S. typhimurium) and Listeria. In one embodiment, the vector of the invention is a bacterial vector and the bacterium is a Gram-negative bacterium. In one embodiment, the vector of the invention is a bacterial vector selected from E. coli vectors, Shigella vectors, Salmonella vectors and Listeria vectors.

Without wishing to be bound by any particular theory, the inventors believe that antigen delivery using the vectors of the invention stimulates, among other responses, a T cell response in a subject. . Accordingly, the inventors believe that one way in which the present invention provides protection against hantavirus infection is by stimulating T cell responses and the cell-mediated immune system. In addition, humoral (antibody)-based protection can also be achieved.

A viral vector of the invention may be a non-replicating viral vector.

As used herein, a non-replicating viral vector is a viral vector that lacks the ability to productively replicate following infection of a target cell. Thus, the ability of a non-replicating viral vector to produce a copy of itself following infection of a target cell (e.g., human target cells of an individual vaccinated with a non-replicating viral vector) is highly diminished or non-existent. . Such viral vectors are sometimes referred to as attenuated or replication defective. The cause may be the loss/deletion of genes essential for replication in the target cell. Therefore, non-replicating viral vectors cannot effectively produce copies of themselves after infection of target cells. Non-replicating viral vectors may therefore advantageously have an improved safety profile compared to replication-competent viral vectors. A non-replicating viral vector can retain the ability to replicate in cells other than the target cell, allowing production of the viral vector. By way of example, non-replicating viral vectors (e.g., non-replicating poxvirus vectors) lack the ability to productively replicate in target cells such as mammalian cells (e.g., human cells), but avian cells (e.g., chicken cells). may retain the ability to replicate in (embryonic fibroblasts or CEF cells) (thus allowing vector production).

A viral vector of the invention may be a non-replicating poxvirus vector. Thus, in one embodiment, the viral vector encoding hantavirus NP or an antigenic fragment thereof is a non-replicating poxvirus vector.

In one embodiment, the non-replicating poxvirus vector is selected from modified vaccinia virus Ankara (MVA) vectors, NYVAC vaccinia virus vectors, canarypox (ALVAC) vectors, and fowlpox (FPV) vectors. Both MVA and NYVAC are attenuated derivatives of vaccinia virus. Compared to vaccinia virus, MVA lacks approximately 26 of the approximately 200 open reading frames.

In one embodiment, the non-replicating poxvirus vector is an FPV vector.

In preferred embodiments, the non-replicating poxvirus vector is an MVA vector.

A viral vector of the invention may be an adenoviral vector. Thus, in one embodiment, the viral vector encoding hantavirus NP or an antigenic fragment thereof is an adenoviral vector.

In one embodiment, the adenoviral vector is a non-replicating adenoviral vector (non-replicating is defined as above). Adenoviruses can be rendered non-replicating by deletion of the E1 gene region or both the E1 and E3 gene regions. Alternatively, the adenovirus can be rendered non-replicating by modifying the E1 gene region or the E1 and E3 gene regions such that said gene regions are non-functional. For example, a non-replicating adenovirus may lack a functional E1 region, or may lack functional E1 and E3 gene regions. Adenoviruses are thus rendered replication-incompetent in most mammalian cell lines and do not replicate in immunized mammals. Most preferably, deletions of both the E1 and E3 gene regions are present in the adenovirus, thus allowing larger size transgenes to be inserted. This is particularly important for expressing larger antigens, or when expressing multiple antigens in a single vector, or when using large promoter sequences such as the CMV promoter. Deletions of the E3 region as well as the E1 region are particularly preferred for recombinant Ad5 vectors. Optionally, the E4 region can also be manipulated.

In one embodiment, the adenoviral vector is a human adenoviral vector, a simian adenoviral vector, a group B adenoviral vector, a group C adenoviral vector, a group E adenoviral vector, an adenoviral 6 vector, a PanAd3 vector, an adenoviral C3 vector. , ChAdY25 vectors, AdC68 vectors, and Ad5 vectors.

A viral vector of the present invention may be a measles virus vector. Thus, in one embodiment, the viral vector encoding hantavirus NP or an antigenic fragment thereof is a measles virus vector.

In one embodiment, the expression cassette comprising a nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) is less than 9 kb (e.g., 9.0, 8.5, 8.0, 7.5, 7.0). , 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8 , 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3 .5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3 , 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1 .0 kb).

In one embodiment, an expression cassette comprising a nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) is less than 8 kb (e.g., 8.0, 7.9, 7.8, 7.7, 7.6 , 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6 .3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1 , 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3 .8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6 , 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1 .3, 1.2, 1.1, 1.0 kb).

In one embodiment, the expression cassette comprising a nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) is less than 7 kb (e.g., 7.0, 6.9, 6.8, 6.7, 6.6). , 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5 .3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1 , 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2 .8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6 , 1.5, 1.4, 1.3, 1.2, 1.1, less than 1.0 kb).

In one embodiment, the expression cassette comprising a nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) is less than 6 kb (e.g., 6, 5.9, 5.8, 5.7, 5.6, 5 .5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3 , 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3 .0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8 , 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, less than 1.0 kb).

In one embodiment, an expression cassette comprising a nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) is less than 5 kb (e.g., 5.0, 4.9, 4.8, 4.7, 4.6 , 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3 .3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1 , 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, less than 1.0 kb).

In one embodiment, an expression cassette comprising a nucleic acid sequence encoding hantavirus NP (or an antigenic fragment thereof) is less than 4.5 kb (e.g., 4.5, 4.4, 4.3, 4.2, 4 .1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9 , 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1 .6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

In one embodiment where the vector is a viral vector, the virus (ie, viral vector) is not a pseudotyped virus. Thus, in one embodiment, the envelope of the viral vector does not contain foreign glycoproteins (ie, glycoproteins not native to said viral vector).

In one embodiment, where the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding hantavirus NP or an antigenic fragment thereof comprises a nucleic acid sequence encoding a hantavirus glycoprotein.

In one embodiment in which the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding a hantavirus NP or antigenic fragment thereof comprises a nucleic acid sequence encoding an epitope of the hantavirus glycoprotein (GP). .

In one embodiment, where the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequences encoding hantavirus NP or antigenic fragments thereof do not include nucleic acid sequences encoding hantavirus glycoproteins (GP).

In one embodiment in which the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding a hantavirus NP or antigenic fragment thereof comprises a nucleic acid sequence encoding an epitope of the hantavirus glycoprotein (GP). do not have.

In one embodiment, the hantavirus nucleoprotein or antigenic fragment thereof is the only hantavirus nucleic acid sequence in the vector.

In one embodiment, in which the vector is a non-replicating poxvirus vector, the vector is stable, expresses the hantavirus NP product, and induces a protective immune response in the subject.

In one embodiment in which the vector is an adenoviral vector, the vector is stable, expresses the hantavirus NP product, and induces a protective immune response in the subject.

The nucleic acid sequences described above may include nucleic acid sequences encoding hantavirus NP containing fusion proteins. A fusion protein may comprise a hantavirus NP polypeptide fused to one or more additional polypeptides, such as an epitope tag, another antigen, or a protein that increases immunogenicity (eg, flagellin).

In one embodiment, the nucleic acid sequence encoding hantavirus NP (as described above) further encodes a tissue plasminogen activator (tPA) signal sequence and/or a V5 fusion protein sequence. In certain embodiments, the presence of the tPA signal sequence can provide increased immunogenicity and the presence of the V5 fusion protein sequence can provide identification of the expressed protein by immunolabeling.

In one embodiment, the vector (as described above) further comprises a nucleic acid sequence encoding an adjuvant (eg, cholera toxin, E. coli lethal toxin, or flagellin).

In one embodiment, the vector does not contain a nucleic acid sequence encoding an adjuvant. In one embodiment, the vector does not contain a nucleic acid sequence encoding Hsp70.

The bacterial vectors of the invention can be produced by using any technique for manipulating and producing recombinant bacteria known in the art.

In another aspect, the invention provides nucleic acid sequences encoding the above viral vectors. The nucleic acid sequence may thus encode a non-replicating poxvirus vector as described above. Accordingly, the nucleic acid sequence may encode an adenoviral vector as described above.

Nucleic acid sequences encoding viral vectors (as described above) may be produced by using any technique for manipulating and producing recombinant nucleic acids known in the art.

In one aspect, the invention provides a method of preparing a viral vector (as described above) comprising the steps of providing a nucleic acid comprising a nucleic acid sequence encoding the vector (as described above); culturing said host cell under conditions suitable for the growth of said vector; and obtaining said vector from said host cell.

As used herein, "transfection" can mean any non-viral method of introducing nucleic acid into a cell. A nucleic acid can be any nucleic acid suitable for transfecting a host cell. Thus, in one embodiment the nucleic acid is a plasmid. A host cell can be any cell in which a vector (eg, a non-replicating poxvirus vector or adenovirus vector as described above) can be grown. As used herein, "culturing a host cell under conditions suitable for the propagation of the vector" refers to conditions of the art that are suitable for the selected host cell and allow the vector to be produced in said host cell. means using any cell culture conditions and techniques known in the art. As used herein, "obtaining the vector" means using any technique known in the art suitable for separating the vector from the host cell. Thus, the host cell can be lysed to release the vector. The vector can then be isolated and purified using any suitable method or methods known in the art.

In one aspect, the invention provides a host cell comprising a nucleic acid sequence encoding a viral vector as described above. A host cell can be any cell in which a viral vector (eg, a non-replicating poxvirus vector or adenoviral vector as described above) can be grown or propagated. In one embodiment, the host cells are 293 cells (also known as HEK or human embryonic kidney cells), CHO cells (Chinese Hamster Ovary), CCL81.1 cells, Vero cells, HELA cells, Per. C6 cells, BHK cells (baby hamster kidney), primary CEF cells (chicken embryo fibroblasts), duck embryo fibroblasts, DF-1 cells or rat IEC-6 cells.

The present invention also provides compositions comprising the vectors described above.

In one aspect, the invention provides a composition comprising a vector (as described above) and a pharmaceutically acceptable carrier.

Substances suitable for use as pharmaceutically acceptable carriers are known in the art. Non-limiting examples of pharmaceutically acceptable carriers include water, saline and phosphate buffered saline. However, in some embodiments the composition is in lyophilized form, in which case it may contain a stabilizer such as bovine serum albumin (BSA). In some embodiments, it may be desirable to formulate the composition with preservatives, such as thiomersal or sodium azide, to facilitate long-term storage. Examples of buffering agents include, but are not limited to, sodium succinate (pH 6.5), and phosphate buffered saline (PBS; pH 7.4).

In addition to pharmaceutically acceptable carriers, the compositions of the invention can be further combined with one or more of salts, excipients, diluents, adjuvants, immunomodulatory agents and/or antimicrobial compounds.

Advantageously, the vectors of the invention have been demonstrated to provide protective immune responses without the use of adjuvants. Therefore, in one embodiment, the composition of the invention does not contain an adjuvant.

The compositions can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids (eg, hydrochloric acid or phosphoric acid, etc.) or organic acids (eg, acetic acid, oxalic acid, tartaric acid, maleic acid, etc.). Salts formed with free carboxyl groups also include inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylamine. It can be derived from aminoethanol, histidine, procaine, and the like.

In one embodiment, the composition (as described above) further comprises at least one hantavirus NP antigen (ie, an antigen present in the composition in the form of a polypeptide). Thus, a composition can include both a vector and a polypeptide. In one embodiment, the polypeptide antigen is hantavirus NP. In one embodiment, the polypeptide antigen is hantavirus GP. In one embodiment, the presence of the polypeptide antigen means that improved simultaneous T cell and antibody responses can be achieved after administration of the composition to a subject. In one embodiment, the T cell and antibody responses achieved are greater than those achieved using either the vector or the polypeptide antigen alone.

In one embodiment, the polypeptide antigen is not attached to the vector. In one embodiment, the polypeptide antigen is a separate component of the vector. In one embodiment, the polypeptide antigen is provided separately from the vector.

In one embodiment, the polypeptide antigen is a variant of the antigen encoded by the vector. In one embodiment, the polypeptide antigen is a fragment of the antigen encoded by the vector. In one embodiment, the polypeptide antigen comprises at least part of the polypeptide sequence encoded by the nucleic acid sequence of the vector. Thus, a polypeptide antigen may correspond to at least part of an antigen encoded by a vector.

In one embodiment, the polypeptide antigen has an amino acid sequence selected from SEQ ID NOS: 4, 7 and 10 and at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94 , 95, 96, 97, 98, 99 or 100%).

In one embodiment, the polypeptide antigen has an amino acid sequence selected from SEQ ID NOS: 11 and 12 and at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95 , 96, 97, 98, 99 or 100%).

In one embodiment, the polypeptide antigen has an amino acid sequence selected from SEQ ID NOS: 13 and 14 and at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95 , 96, 97, 98, 99 or 100%).

In one embodiment, the polypeptide antigen has an amino acid sequence selected from SEQ ID NOS: 31 and 32 and at least 70% (e.g., at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95 , 96, 97, 98, 99 or 100%).
ＭＡＴＭＥＥＩＱＲＥＩＳＡＨＥＧＱＬＶＩＡＲＱＫＶＫＤＡＥＫＱＹＥＫＤＰＤＤＬＮＫＲＡＬＨＤＲＥＳＶＡＡＳＩＱＳＫＩＤＥＬＫＲＱＬＡＤＲＩＡＡＧＫＮＩＧＱＤＲＤＰＴＧＶＥＰＧＤＨＬＫＥＲＳＡＬＳＹＧＮＴＬＤＬＮＳＬＤＩＤＥＰＴＧＱＴＡＤＷＬＴＩＩＶＹＬＴＳＦＶＶＰＩＩＬＫＡＬＹＭＬＴＴＲＧＲＱＴＳＫＤＮＫＧＭＲＩＲＦＫＤＤＳＳＹＥＤＶＮＧＩＲＫＰＫＨＬＹＶＳＭＰＮＡＱＳＳＭＫＡＥＥＩＴＰＧＲＦＲＴＡＶＣＧＬＹＰＡＱＩＫＡＲＮＭＶＳＰＶＭＳＶＶＧＦＬＡＬＡＫＤＷＴＳＲＩＥＥＷＬＧＡＰＣＫＦＭＡＥＳＰＩＡＧＳＬＳＧＮＰＶＮＲＤＹＩＲＱＲＱＧＡＬＡＧＭＥＰＫＥＦＱＡＬＲＱＨＳＫＤＡＧＣＴＬＶＥＨＩＥＳＰＳＳＩＷＶＦＡＧＡＰＤＲＣＰＰＴＣＬＦＶＧＧＭＡＥＬＧＡＦＦＳＩＬＱＤＭＲＮＴＩＭＡＳＫＴＶＧＴＡＤＥＫＬＲＫＫＳＳＦＹＱＳＹＬＲＲＴＱＳＭＧＩＱＬＤＱＲＩＩＶＭＦＭＶＡＷＧＫＥＡＶＤＮＦＨＬＧＤＤＭＤＰＥＬＲＳＬＡＱＩＬＩＤＱＫＶＫＥＩＳＮＱＥＰＭＫＬＭＬＳＹＧＮＶＬＤＬＮＨＬＤＩＤＥＰＴＧＱＴＡＤＷＬＧＩＶＩＹＬＴＳＦＶＶＰＩＬＬＫＡＬＹＭＬＴＴＲＧＲＱＴＴＫＤＮＫＧＴＲＩＲＦＫＤＤＳＳＦＥＤＶＮＧＩＲＫＰＫＨＬＹＶＳＬＰＮＡＱＳＳＭＫＡＥＥＩＴＰＧＲＹＲＴＡＩＣＧＬＹＰＡＱＩＫＡＲＱＭＩＳＰＶＭＳＶＩＧＦＬＡＬＡＫＤＷＳＤＲＩＥＱＷＬＳＥＰＣＫＬＬＰＤＴＡＡＶＳＬＬＧＧＰＡＴＮＲＤＹＬＲＱＲＱＶＡＬＧＮＭＥＴＫＥＳＫＡＩＲＱＨＡＥＡＡＧＣＳＭＩＥＤＩＥＳＰＳＳＩＷＶＦＡＧＡＰＤＲＣＰＰＴＣＬＦＩＡＧＭＡＥＬＧＡＦＦＳＩＬＱＤＭＲＮＴＩＭＡＳＫＴＶＧＴＳＥＥＫＬＲＫＫＳＳＦＹＱＳＹＬＲＲＴＱＳＭＧＩＱＬＤＱＲＩＩＶＬＦＭＶＡＷＧＫＥＡＶＤＮＦＨＬＧＤＤＭＤＰＥＬＲＴＬＡＱＳＬＩＤＶＫＶＫＥＩＳＮＱＥＰＬＫＬ（配列番号３１）
ＭＬＳＹＧＮＶＬＤＬＮＨＬＤＩＤＥＰＴＧＱＴＡＤＷＬＧＩＶＩＹＬＴＳＦＶＶＰＩＬＬＫＡＬＹＭＬＴＴＲＧＲＱＴＴＫＤＮＫＧＴＲＩＲＦＫＤＤＳＳＦＥＤＶＮＧＩＲＫＰＫＨＬＹＶＳＬＰＮＡＱＳＳＭＫＡＥＥＩＴＰＧＲＹＲＴＡＩＣＧＬＹＰＡＱＩＫＡＲＱＭＩＳＰＶＭＳＶＩＧＦＬＡＬＡＫＤＷＳＤＲＩＥＱＷＬＳＥＰＣＫＬＬＰＤＴＡＡＶＳＬＬＧＧＰＡＴＮＲＤＹＬＲＱＲＱＶＡＬＧＮＭＥＴＫＥＳＫＡＩＲＱＨＡＥＡＡＧＣＳＭＩＥＤＩＥＳＰＳＳＩＷＶＦＡＧＡＰＤＲＣＰＰＴＣＬＦＩＡＧＭＡＥＬＧＡＦＦＳＩＬＱＤＭＲＮＴＩＭＡＳＫＴＶＧＴＳＥＥＫＬＲＫＫＳＳＦＹＱＳＹＬＲＲＴＱＳＭＧＩＱＬＤＱＲＩＩＶＬＦＭＶＡＷＧＫＥＡＶＤＮＦＨＬＧＤＤＭＤＰＥＬＲＴＬＡＱＳＬＩＤＶＫＶＫＥＩＳＮＱＥＰＬＫＬＭＡＴＭＥＥＩＱＲＥＩＳＡＨＥＧＱＬＶＩＡＲＱＫＶＫＤＡＥＫＱＹＥＫＤＰＤＤＬＮＫＲＡＬＨＤＲＥＳＶＡＡＳＩＱＳＫＩＤＥＬＫＲＱＬＡＤＲＩＡＡＧＫＮＩＧＱＤＲＤＰＴＧＶＥＰＧＤＨＬＫＥＲＳＡＬＳＹＧＮＴＬＤＬＮＳＬＤＩＤＥＰＴＧＱＴＡＤＷＬＴＩＩＶＹＬＴＳＦＶＶＰＩＩＬＫＡＬＹＭＬＴＴＲＧＲＱＴＳＫＤＮＫＧＭＲＩＲＦＫＤＤＳＳＹＥＤＶＮＧＩＲＫＰＫＨＬＹＶＳＭＰＮＡＱＳＳＭＫＡＥＥＩＴＰＧＲＦＲＴＡＶＣＧＬＹＰＡＱＩＫＡＲＮＭＶＳＰＶＭＳＶＶＧＦＬＡＬＡＫＤＷＴＳＲＩＥＥＷＬＧＡＰＣＫＦＭＡＥＳＰＩＡＧＳＬＳＧＮＰＶＮＲＤＹＩＲＱＲＱＧＡＬＡＧＭＥＰＫＥＦＱＡＬＲＱＨＳＫＤＡＧＣＴＬＶＥＨＩＥＳＰＳＳＩＷＶＦＡＧＡＰＤＲＣＰＰＴＣＬＦＶＧＧＭＡＥＬＧＡＦＦＳＩＬＱＤＭＲＮＴＩＭＡＳＫＴＶＧＴＡＤＥＫＬＲＫＫＳＳＦＹＱＳＹＬＲＲＴＱＳＭＧＩＱＬＤＱＲＩＩＶＭＦＭＶＡＷＧＫＥＡＶＤＮＦＨＬＧＤＤＭＤＰＥＬＲＳＬＡＱＩＬＩＤＱＫＶＫＥＩＳＮＱＥＰＭＫＬ（配列番号３２）

The polypeptide antigen can be the same (or similar) to that encoded by the nucleic acid sequence of the vector of the composition. Thus, administration of a composition comprising a vector and a polypeptide antigen can be used to achieve an enhanced immune response to a single antigen, said enhanced immune response combined as described above. including T cell and antibody responses.

In one embodiment, the composition (as described above) of the invention comprises at least one naked DNA encoding hantavirus NP or an antigenic fragment thereof (i.e., not part of the viral vector of the invention, but with it). separate DNA molecules). In one embodiment, the naked DNA comprises a nucleic acid sequence selected from SEQ ID NOS: 1, 2, 3, 5, 6, 8, 9 and 15-30 and at least 70% (e.g. , 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%). In one embodiment, the naked DNA comprises an amino acid sequence selected from SEQ ID NOS: 4, 7, 10-14, 31 and 32 and at least 70% (eg, at least 70, 75, 80, 82, 84, 86, 88 , 90, 92, 94, 95, 96, 97, 98, 99 or 100%).

In one embodiment, the composition of the invention (as described above) further comprises an adjuvant. Non-limiting examples of adjuvants suitable for use with the compositions of the invention include aluminum phosphate, aluminum hydroxide and related compounds; monophosphoryl lipid A and related compounds; outer membrane vesicles from bacteria; liposomal adjuvants such as virosomes, Freund's adjuvant and related mixtures; polylactide-co-glycolide acid (PLGA) particles; cholera toxin; E. coli lethal toxin; and flagellin.

The vectors and compositions (as described above) of the invention can be used as vaccines. A composition of the invention may therefore be a vaccine composition.

As used herein, a vaccine when administered to an animal subject, such as a mammal (e.g., a human, bovine, porcine, ovine, goat, equine, deer, canine or feline subject; particularly a human subject) , is a formulation that stimulates protective immune responses against infectious diseases. The immune response can be a humoral and/or cell-mediated immune response. Vaccines may therefore stimulate B cells and/or T cells.

The term "vaccine" is used herein interchangeably with the terms "therapeutic/prophylactic composition," "immunogenic composition," "formulation," "antigenic composition," or "medicament." used.

In one aspect, the invention provides a vector (as described above) or a composition (as described above) for use in medicine.

In one aspect, the invention provides a vector (as described above) or composition (as described above) for use in a method of inducing an immune response in a subject. The immune response can be to hantavirus antigens (eg, hantavirus NP) and/or to hantavirus infection. Accordingly, the vectors and compositions of the invention can be used (eg, as immunogenic compositions or as vaccines) to induce an immune response against hantavirus NP in a subject.

In one embodiment the immune response comprises a T cell response.

In one embodiment, a method of inducing an immune response in a subject comprises administering to the subject an effective amount of a vector (as described above) or a composition (as described above).

In one aspect, the invention provides a vector (as described above) or composition (as described above) for use in a method of preventing or treating hantavirus infection in a subject.

In one embodiment, the invention provides a vector (as above) or composition (as above) for use in a method of preventing or treating HFRS in a subject.

The vectors and compositions of the present invention are ideally suited for use in the prevention or treatment of HFRS, especially when the hantavirus nucleoprotein or antigenic fragment thereof is derived from Seoul virus. As noted above, Seoul virus is typically associated with the cause of HFRS.

The vectors and compositions of the invention are ideally suited for use in the prevention or treatment of HFRS, especially when the hantavirus nucleoprotein or antigenic fragment thereof is derived from hantavirus. As noted above, Hantan virus is typically associated with the cause of HFRS.

The vectors and compositions of the present invention are particularly useful for hantavirus nucleoproteins or antigenic fragments thereof, such as Seoul virus nucleoproteins (or antigenic fragments thereof) and hantavirus nucleoproteins (or antigenic fragments thereof), as demonstrated in the Examples. or an antigenic fragment thereof) are ideally suited for use in the prevention or treatment of HFRS.

As used herein, the term "preventing" includes preventing the onset of hantavirus infection and/or reducing the severity of the intensity of hantavirus infection. Thus, "preventing" includes vaccination.

As used herein, the term "treating" encompasses therapeutic and prophylactic/prophylactic measures (including post-exposure prophylaxis), including post-infection therapy of hantavirus infection and amelioration thereof. .

In one embodiment, the hantavirus infection is Seoul virus infection. In one embodiment, the hantavirus infection is a hantavirus infection. In one embodiment, the hantavirus infection is Seoul virus infection and/or Hantavirus infection.

Each of the above methods can comprise administering to the subject an effective amount, eg, a therapeutically effective amount, of the vector or composition of the invention.

In this regard, as used herein, an effective amount is a dose or amount sufficient to achieve a desired biological result. As used herein, a therapeutically effective amount means, upon single or multiple administrations to a subject (e.g., a mammalian subject, particularly a human subject), more than would be expected in the absence of such treatment. , an amount effective to treat, prevent, suppress, cure, delay, lessen the severity of, ameliorate at least one symptom of the disorder or recurrence of the disorder, or prolong survival of the subject.

Accordingly, the amount of active ingredient administered will depend on the subject to be treated, the ability of the subject's immune system to generate a protective immune response, and the degree of protection required. Precise amounts of active ingredient required for administration will depend on the judgment of the practitioner and may be peculiar to each subject.

Administering to a subject can comprise administering a vector (as described above) or a composition (as described above) to a subject, wherein the composition is administered multiple times sequentially (e.g., the composition is administered 2, 3 or 4 times). Thus, in one embodiment, a subject is administered a vector (as described above) or a composition (as described above) and then the same vector or composition (or a substantially similar vector or composition) is administered to a different Dosing again at time points.

In one embodiment, administering to a subject comprises administering a vector (as described above) or a composition (as described above) to a subject, wherein said composition comprises the substance of another immunogenic composition. administered prior to, concurrently with, or subsequently.

Prior, concurrent, and sequential dosing regimens are discussed in more detail below.

In certain embodiments, the above methods further comprise administering to the subject a second vector comprising a nucleic acid sequence encoding hantavirus NP. Preferably, the second vector is a vector of the invention as described above (such as a viral vector, eg a non-replicating poxvirus vector or an adenoviral vector as described above).

In one embodiment, the first and second vectors are of the same vector type. In one embodiment, the first and second vectors are different vector types. In one embodiment, the first vector is an adenoviral vector (as described above) and the second vector is a non-replicating poxvirus vector (as described above). In one embodiment, the first vector is a non-replicating poxvirus vector (as described above) and the second vector is an adenovirus vector (as described above).

In one embodiment, the first and second vectors are administered sequentially in any order. Thus, the first (“1”) and second (“2”) vectors can be administered to the subject in a 1 to 2 order or 2 to 1 order.

"Continuously administered" as used herein has the meaning of "continuous administration" as defined below. Thus, the first and second vectors are administered sequentially at (substantially) different times.

In one embodiment, the first and second vectors are administered as part of a prime-boost administration protocol. Thus, a first vector can be administered to a subject as a "prime" and a second vector can be subsequently administered to the same subject as a "boost". The prime-boost protocol is described below.

In one embodiment, each of the above methods further comprises administering a hantavirus polypeptide antigen to the subject. In one embodiment, the hantavirus polypeptide antigen is hantavirus NP (or an antigenic fragment thereof) as described above. In one embodiment, the hantavirus polypeptide antigen has an amino acid sequence selected from SEQ ID NOS: 4, 7, 10-14, 31 and 32 and at least 70% (eg, at least 70, 75, 80, 82, 84, 86 , 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%).

In one embodiment, the polypeptide antigen is administered separately from administration of the vector, preferably said polypeptide antigen and vector are administered sequentially. In one embodiment, the vector (“V”) and polypeptide antigen (“P”) may be administered in V to P or P to V order.

In one embodiment, each of the above methods further comprises administering to the subject naked DNA encoding hantavirus NP or an antigenic fragment thereof. In one embodiment, the naked DNA comprises a nucleic acid sequence selected from SEQ ID NOS: 1, 2, 3, 5, 6, 8, 9 and 15-30 and at least 70% (e.g. , 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%). In one embodiment, the naked DNA comprises an amino acid sequence selected from SEQ ID NOS: 4, 7, 10-14, 31 and 32 and at least 70% (eg, at least 70, 75, 80, 82, 84, 86, 88 , 90, 92, 94, 95, 96, 97, 98, 99 or 100%).

In one embodiment, the naked DNA is administered separately from administration of the vector, preferably the naked DNA and vector are administered sequentially. In one embodiment, the vector (“V”) and naked DNA (“D”) can be administered in the order V to D or in the order D to V.

In one embodiment, naked DNA (as described above) is administered as part of a prime-boost protocol.

Heterologous prime-boost approaches can improve immune responses by allowing repeated vaccinations without increasing anti-vector immunity. Hantavirus NP or antigenic fragments thereof can be sequentially delivered via different vectors (as described above) or naked DNA vectors (as described above). Any heterologous prime-boost vaccination regimen will increase NP-specific antibody responses, increase NP-specific T cell responses, and/or reduce clinical disease compared to the use of a single vector. Suitable combinations of vectors include, but are not limited to:
DNA prime, MVA boost DNA prime, fowlpox boost Fowlpox prime, MVA boost MVA prime, fowlpox boost DNA prime, fowlpox boost, MVA boost MVA prime, adenovirus boost

As used herein, the term polypeptide includes peptides and proteins.

In certain embodiments, the above methods further comprise administering an adjuvant to the subject. An adjuvant can be administered with one, two, three, or all four of the first vector, second vector, polypeptide antigen, and naked DNA.

The immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g., vaccines) of the invention are given on a single dose schedule (i.e., the entire dose is given substantially at once). obtain. The immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (eg, vaccines) of the invention may be given on a multiple dose schedule.

A multiple dose schedule may be from 1 to 6 individual doses in the primary course of treatment (e.g., vaccination), with subsequent time intervals required to maintain and/or enhance the immune response, e.g. (For human subjects), a schedule of 1-4 months for the second dose followed by another dose, optionally followed by another 1-4 months later.

Dosage regimens are determined, at least in part, by individual needs and depend on the judgment of the practitioner (eg, physician or veterinarian).

Simultaneous administration means administration at (substantially) the same time.

Sequential administration of two or more compositions/therapeutic agents/vaccine means that the compositions/therapeutic agents/vaccine are administered sequentially at (substantially) different times.

For example, sequential administration may include two or more doses at different times separated by several days (eg, at least 1, 2, 5, 10, 15, 20, 30, 60, 90, 100, 150 or 200 days). Administration of compositions/therapeutic agents/vaccine may be included.

For example, in one embodiment, vaccines of the invention may be administered as part of a "prime-boost" vaccination regimen.

In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (eg, vaccines) of the present invention are, for example, immunoglobulins, antibiotics, interleukins (eg, IL-2, IL-12), and/or cytokines (eg, IFNγ) in combination (simultaneously or sequentially) with one or more immunomodulatory agents selected from mammals (eg, human, bovine, porcine, ovine, goats, horses, deer, bears, canine or feline subjects).

Immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (eg, vaccines) contain 5% to 95% active ingredient, such as at least 10% or 25% active ingredient, or at least 40% It may contain active ingredient, or at least 50, 55, 60, 70 or 75% active ingredient.

Immunogenic compositions, therapeutic formulations, pharmaceuticals, pharmaceutical compositions and prophylactic formulations (e.g., vaccines) are administered in a manner compatible with the dosage formulation, and in such amount as is prophylactically and/or therapeutically effective. be.

Administration of immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions and prophylactic formulations (eg vaccines) is generally by conventional routes such as intravenous, subcutaneous, intraperitoneal or mucosal routes. Administration can be parenteral, eg, by subcutaneous or intramuscular injection.

Immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (eg, vaccines) of the invention can be prepared for injection, either as liquid solutions or suspensions. Alternatively, solid forms suitable for solution in, or suspension in, liquid prior to injection may be prepared. The preparation may also be emulsified, or the peptide may be encapsulated in liposomes or microcapsules.

The active ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In addition, immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g., vaccines), if desired, may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, and/or pH buffering agents. can contain

Generally, the carrier is a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, saline and phosphate buffered saline. However, in some embodiments the composition is in lyophilized form, in which case it may contain a stabilizer such as bovine serum albumin (BSA). In some embodiments, it may be desirable to formulate the composition with preservatives, such as thiomersal or sodium azide, to facilitate long-term storage.

Examples of buffering agents include, but are not limited to, sodium succinate (pH 6.5), and phosphate buffered saline (PBS; pH 6.5 and 7.5).

Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations or formulations suitable for distribution as an aerosol. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides, and such suppositories contain 0.5%-10%, preferably 1%-2%. It can be formed from a mixture containing a range of active ingredients.

Oral formulations include commonly used excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

It may be desirable to direct a composition of the invention (as described above) to the respiratory system of the subject. Efficient delivery of therapeutic/prophylactic compositions or medicaments to the pulmonary infected site can be achieved by oral or intranasal administration.

Formulations for intranasal administration may be in the form of nasal drops or nasal spray. Intranasal formulations may comprise droplets having an approximate diameter in the range 100-5000 μm, such as 500-4000 μm, 1000-3000 μm or 100-1000 μm. Alternatively, in terms of volume, droplets may range from about 0.001-100 μl, such as 0.1-50 μl or 1.0-25 μl, or such as 0.001-1 μl.

Alternatively, the therapeutic/prophylactic formulation or medicament may be an aerosol formulation. Aerosol formulations can take the form of powders, suspensions or solutions. Aerosol particle size is related to the delivery capacity of the aerosol. Smaller particles may travel further down the respiratory airways toward the alveoli than larger particles. In one embodiment, the aerosol particles have a diameter distribution to facilitate delivery along the length of bronchi, bronchioles, and alveoli. Alternatively, the particle size distribution can be selected to target specific portions of the respiratory airways, such as the alveoli. For aerosol delivery of pharmaceuticals, the particles may have diameters in the approximate range of 0.1-50 μm, preferably 1-25 μm, more preferably 1-5 μm.

Aerosol particles may be for delivery using a nebulizer (eg, through the mouth) or nasal spray. Aerosol formulations may optionally contain a propellant and/or a surfactant.

In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g., vaccines) of the invention are formulated with pharmaceutically acceptable carriers and, optionally, salts, excipients. , diluents and/or adjuvants.

In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (eg, vaccines) of the invention are, for example, immunoglobulins, antibiotics, interleukins (eg, IL-2). , IL-12), and/or cytokines (eg, IFNγ).

The present invention includes polypeptides substantially homologous to polypeptides based on any one of the polypeptide antigens (including fragments thereof) identified in this application. The terms "sequence identity" and "sequence homology" are considered synonymous herein.

By way of example, a polypeptide of interest has at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity with the amino acid sequence of a reference polypeptide. can comprise an amino acid sequence having

There are many established algorithms available for aligning two amino acid sequences. Typically, one sequence acts as a reference sequence, to which test sequences can be compared. The sequence comparison algorithm calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the specified program parameters. Alignment of amino acid sequences for comparison can be performed, for example, by computer-implemented algorithms (eg, GAP, BESTFIT, FASTA or TFASTA), or the BLAST and BLAST 2.0 algorithms.

The BLOSUM62 table shown below is an amino acid substitution matrix derived from approximately 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of over 500 groups of related proteins (Henikoff & Henikoff, Proc. USA 89:10915-10919, 1992; incorporated herein by reference). Amino acids are indicated by the standard single-letter code. Percent identity is calculated as follows.
Total number of identical matches ___________________ x100
[length of longer sequence + align two sequences
number of gaps introduced into the longer sequence due to]

Table of BLOSUM62
ARNDCQEGHILKMFPSTWYV
A4
R-1 5
N-206
D -2 -2 1 6
C0-3-3-39
Q -1 1 0 0 -3 5
E -1 0 0 2 -4 2 5
G 0 -2 0 -1 -3 -2 -2 6
H -2 0 1 -1 -3 0 0 -2 8
I -1 -3 -3 -3 -1 -3 -3 -4 -3 4
L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4
K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5
M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5
F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6
P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7
S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4
T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5
W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11
Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7
V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4

In homology comparisons, identity is measured at least 10 amino acid residues long (e.g. , 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550 or 570 amino acid residues long—e.g., the full length of the maximum reference sequence can exist over

A substantially homologous polypeptide has one or more amino acid substitutions, deletions or additions. In many embodiments, these changes are minor in nature, eg, involving only conservative amino acid substitutions. Conservative substitutions are substitutions made by replacing one amino acid in the following group with another amino acid: basic: arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; polar: glutamine, asparagine. hydrophobic: leucine, isoleucine, valine; aromatic: phenylalanine, tryptophan, tyrosine; small molecules: glycine, alanine, serine, threonine, methionine. Substantially homologous polypeptides include other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically 1 to about 30 amino acids (eg, 1-10 or 1-5 amino acids) ); and small amino- or carboxyl-terminal extensions, such as amino-terminal methionine residues, small linker peptides of up to about 20-25 residues, or polypeptides containing affinity tags.

As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably and do not imply any length limitation. As used herein, the terms "nucleic acid" and "nucleotide" are used interchangeably. The terms "nucleic acid sequence" and "polynucleotide" encompass DNA (including cDNA) and RNA sequences.

The polynucleotide sequences of the present invention include nucleic acid sequences removed from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogs or analogs synthesized biologically by heterologous systems. including.

Polynucleotides of the invention may be prepared by any means known in the art. For example, large amounts of polynucleotide can be produced by replication in a suitable host cell. A natural or synthetic DNA segment encoding a desired segment is incorporated into a recombinant nucleic acid construct, typically a DNA construct, capable of introduction into and replication in prokaryotic or eukaryotic cells. DNA constructs are generally suitable for autonomous replication in unicellular hosts such as yeast or bacteria, although introduction and integration into the genome of cultured insect, mammalian, plant or other eukaryotic cell lines is also contemplated. obtain.

A polynucleotide of the invention may also be produced by chemical synthesis, such as the phosphoramidite method or the triester method, performed on commercially available automated oligonucleotide synthesizers. Double-stranded fragments are prepared from a single-stranded product of chemical synthesis by synthesizing complementary strands and annealing the strands together under appropriate conditions, or using DNA polymerase with appropriate primer sequences. It can be obtained by adding a complementary strand.

The term "isolated" in the context of the present invention, when applied to nucleic acid sequences, means that the polynucleotide sequence has been removed from its natural genetic environment and thus free of other exogenous or undesired coding sequences. It does not contain (although it may contain naturally occurring 5' and 3' untranslated regions such as promoters and terminators), indicating a form suitable for use within genetically engineered protein production systems. Such isolated molecules are molecules that are separated from their natural environment.

Considerable sequence variation among the polynucleotides of the invention is possible given the degeneracy of the genetic code. Degenerate codons that encompass all possible codons for a given amino acid are shown below:

Those skilled in the art will appreciate that flexibility exists when determining degenerate codons to represent all possible codons encoding each amino acid. For example, some polynucleotides encompassed by a degenerate sequence may encode variant amino acid sequences, although those skilled in the art readily identify such variant sequences by reference to the amino acid sequences of the present invention. be able to.

A "variant" nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or fragment thereof). A nucleic acid sequence or fragment thereof, when optimally aligned (with appropriate nucleotide insertions or deletions) with other nucleic acids (or complementary strands thereof), comprises at least about 70%, 75%, 80% of the nucleotide bases, A reference sequence is "substantially homologous" (or " “substantially the same”). Methods for determining nucleic acid sequence homology are known in the art.

Alternatively, a "variant" nucleic acid sequence may be a reference sequence (or fragment thereof) if the "variant" and the reference sequence are capable of hybridizing under stringent (e.g., highly stringent) hybridization conditions. ) is substantially homologous (or substantially identical to). As will be readily understood by those of skill in the art, nucleic acid sequence hybridization is measured by salt concentration (e.g. NaCl), temperature , or by conditions such as organic solvents. Preferably, stringent temperature conditions are used, for example temperatures generally above 30°C, typically above 37°C, preferably above 45°C. Stringent salt conditions are usually less than 1000 mM, typically less than 500 mM, preferably less than 200 mM. The pH is typically between 7.0 and 8.3. A combination of parameters is much more important than any single parameter.

Methods for determining percent nucleic acid sequence identity are known in the art. As an example, when assessing nucleic acid sequence identity, a sequence having a defined number of contiguous nucleotides is aligned with a nucleic acid sequence (having the same number of contiguous nucleotides) from the corresponding portion of the nucleic acid sequence of the invention. can be made Tools known in the art for determining percent nucleic acid sequence identity include Nucleotide BLAST.

One skilled in the art will recognize that different species exhibit "preferred codon usage". As used herein, the term "preferred codon usage" refers to the codons that are used most frequently in cells of a particular species, and thus one or more of the possible codons encoding each amino acid. Support minority representation. For example, the amino acid threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT, although in mammalian host cells ACC is the most commonly used codon, and in other species a different Thr Codons can be preferred. Preferred codons for a particular host cell type can be introduced into the polynucleotides of the invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance protein production by making protein translation more efficient within a particular cell type or species.

Therefore, in one embodiment of the invention, the nucleic acid sequence is codon-optimized for expression in the host cell.

A "fragment" of a polynucleotide of interest comprises a stretch of contiguous nucleotides from the sequence of said full-length polynucleotide. By way of example, a "fragment" of a polynucleotide of interest is at least 30 contiguous nucleotides from a sequence of said polynucleotide (e.g., at least 35, 50, 75, 100, 150, 200, 250, 300 , 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 , 1600, 1650, 1700 or 1710 contiguous nucleic acid residues). Fragments may contain at least one antigenic determinant and/or may encode at least one antigenic epitope of the corresponding polypeptide of interest and/or may have common antigenic cross-reactivity and/or with the polypeptide of interest. They may have substantially the same in vivo biological activity.

Figures 1A-B show an exemplary MVA vector construction. FIG. 1A provides a schematic of the cassette “MVAHantaNP”. FIG. 1B provides a schematic of plasmid 17ACNHBP_MVA-SEOV-HNT-NP_pMS-RQ (pMVAHantaNP).

FIG. 2 shows PCR confirmation of the pure recombinant nuclear protein—an agarose gel confirming the presence of the MVAHantaNP construct. Flanking primer to flanking primers (SEQ ID NOS: 46 and 47) spanned the entire insert and migrated from the MVA flanking regions on both ends of the vaccine insert to give the expected amplicon size. The contents of the wells are as follows (numbered from left to right):1. ladder;2. positive control (MVA-HantaNP plasmid) GFP to flanking primers - expected size 3260 bp;3. 4. MVA-HantaNP "Batch 1"; 4. MVA-HantaNP "batch 2+3"; 6. MVA-HantaNP "Batch 4+5+6"; 7. Negative control; 8. ladder; 8. Flanking primers from positive control (MVA-HantaNP plasmid) flanking primers - expected size 3788 bp; 10. MVA-HantaNP "Batch 1"; 11. MVA-HantaNP "batch 2+3"; 12. MVA-HantaNP "Batch 4+5+6"; 13. Negative control; ladder

Figure 3 shows a Western blot confirming the expression of the NP/Flag tag. The predicted size of the protein is 89 kDa. The contents of the wells are as follows (numbered from left to right):1. ladder;2. "Passage 3" P3 (1.1.1);3. P3 (4.1.1);4. P3 (4.1.2);5. vaccine batch 1;6. 7. Combination of vaccine batches 2+3; Combination of vaccine batches 4+5+6.

Figure 4 shows clinical scores (% weight gain per day) during the immunization trial. (a) Body weight and (b) body temperature of mice after prime (first arrow, day 0) and boost (second arrow, day 14) immunizations.

Figure 5 shows total ELISPOT responses from vaccinated and non-vaccinated mice.

FIG. 6 shows splenocyte IFN-γ ELISPOT restimulation responses to individual peptide pools (“NP1”-“NP11”). i) Group 1 represents mice prime and boost vaccinated with MVA-HantaNP; ii) Group 2 represents mice vaccinated with a single dose of MVA-HantaNP; iii) Group 3 represents empty MVA. Wild type prime and boost vaccinated mice are shown; iv) Group 4 is PBS control prime and boost.

Figure 7 shows IgG responses to hantavirus NP in mouse serum. Absorbance readings provide a readout of antibody binding activity to recombinant hantavirus NP.

FIG. 8 shows body weight and body temperature of mice after intramuscular (left column) or intranasal (right column) challenge with hantavirus.

Figure 9 shows virus in blood, lungs, kidneys, spleens and livers of mice (a) 5 days after intramuscular challenge; (b) 5 days after intranasal challenge; (c) 14 days after intranasal challenge. indicate quantity.

FIG. 10 shows viral load in kidney, lung and spleen of mice 5 days after intranasal challenge. Results for immunization with empty MVA wild-type vector are represented by circles; results for immunization with MVA-HantaNP are represented by triangles.

Example 1
Example Preparation of MVA-NP (Nucleoprotein) Vector A cassette for MVAHantaNP (denoted "MVAHantaNP") was added to the P11 promoter, green fluorescent protein (GFP) and MH5 promoters, followed by the Kozak sequence upstream of the NP sequence. Prepared by GeneArt (Thermofisher) to contain The nucleoprotein sequence is a chimeric sequence comprising two different sequences of Seoul and Hantan. Downstream is a 24-residue linker sequence followed by a Flag tag epitope and a stop codon. A schematic diagram of MVAHantaNP is provided in FIG. 1(A).

The cassette was inserted into the Sfil/Sfil cloning sites of plasmid pMS-RQ-Bb to prepare plasmid 17ACNHBP_MVA-SEOV-HNT-NP_pMS-RQ (pMVAHantaNP).

A schematic of pMVAHantaNP is provided in FIG. 1(B) and the nucleic acid sequence of pMVAHantaNP is provided in SEQ ID NO:33.
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pMVAHantaNP contains:

DelIII left flanking region:
ＧＴＴＧＧＴＧＧＴＣＧＣＣＡＴＧＧＡＴＧＧＴＧＴＴＡＴＴＧＴＡＴＡＣＴＧＴＣＴＡＡＡＣＧＣＧＴＴＡＧＴＡＡＡＡＣＡＴＧＧＣＧＡＧＧＡＡＡＴＡＡＡＴＣＡＴＡＴＡＡＡＡＡＡＴＧＡＴＴＴＣＡＴＧＡＴＴＡＡＡＣＣＡＴＧＴＴＧＴＧＡＡＡＡＡＧＴＣＡＡＧＡＡＣＧＴＴＣＡＣＡＴＴＧＧＣＧＧＡＣＡＡＴＣＴＡＡＡＡＡＣＡＡＴＡＣＡＧＴＧＡＴＴＧＣＡＧＡＴＴＴＧＣＣＡＴＡＴＡＴＧＧＡＴＡＡＴＧＣＧＧＴＡＴＣＣＧＡＴＧＴＡＴＧＣＡＡＴＴＣＡＣＴＧＴＡＴＡＡＡＡＡＧＡＡＴＧＴＡＴＣＡＡＧＡＡＴＡＴＣＣＡＧＡＴＴＴＧＣＴＡＡＴＴＴＧＡＴＡＡＡＧＡＴＡＧＡＴＧＡＣＧＡＴＧＡＣＡＡＧＡＣＴＣＣＴＡＣＴＧＧＴＧＴＡＴＡＴＡＡＴＴＡＴＴＴＴＡＡＡＣＣＴＡＡＡＧＡＴＧＣＣＡＴＴＣＣＴＧＴＴＡＴＴＡＴＡＴＣＣＡＴＡＧＧＡＡＡＧＧＡＴＡＧＡＧＡＴＧＴＴＴＧＴＧＡＡＣＴＡＴＴＡＡＴＣＴＣＡＴＣＴＧＡＴＡＡＡＧＣＧＴＧＴＧＣＧＴＧＴＡＴＡＧＡＧＴＴＡＡＡＴＴＣＡＴＡＴＡＡＡＧＴＡＧＣＣＡＴＴＣＴＴＣＣＣＡＴＧＧＡＴＧＴＴＴＣＣＴＴＴＴＴＴＡＣＣＡＡＡＧＧＡＡＡＴＧＣＡＴＣＡＴＴＧＡＴＴＡＴＴＣＴＣＣＴＧＴＴＴＧＡＴＴＴＣＴＣＴＡＴＣＧＡＴＧＣＧＧＣＡＣＣＴＣＴＣＴＴＡＡＧＡＡＧＴＧＴＡＡＣＣＧＡＴＡＡＴＡＡＴＧＴＴＡＴＴＡＴＡＴＣＴＡＧＡＣＡＣＣＡＧＣＧＴＣＴＡＣＡＴＧＡＣＧＡＧＣＴＴＣＣＧＡＧＴＴＣＣＡＡＴＴＧＧＴＴＣＡＡＧＴＴＴＴＡＣＡＴＡＡＧＴＡＴＡＡＡＧＴＣＣＧＡＣＴＡＴＴＧＴＴＣＴＡＴＡＴＴＡＴＡＴＡＴＧＧＴＴＧＴＴＧＡＴＧＧＡＴＣＴＧＴＧＡＴＧＣＡＴＧＣＡＡＴＡＧＣＴＧＡＴＡＡＴＡＧＡＡＣＴＴＡＣＧＣＡＡＡＴＡＴＴＡＧＣＡＡＡＡＡＴＡＴＡＴＴＡＧＡＣＡＡＴＡＣＴＡＣＡＡＴＴＡＡＣＧＡＴＧＡＧＴＧＴＡＧＡＴＧＣＴＧＴＴＡＴＴＴＴＧＡＡＣＣＡＣＡＧＡＴＴＡＧＧＡＴＴＣＴＴＧＡＴＡＧＡＧＡＴＧＡＧＡＴＧＣＴＣＡＡＴＧＧＡＴＣＡＴＣＧＴＧＴＧＡＴＡＴＧＡＡＣＡＧＡＣＡＴＴＧＴＡＴＴＡＴＧＡＴＧＡＡＴＴＴＡＣＣＴＧＡＴＧＴＡＧＧＣＧＡＡＴＴＴＧＧＡＴＣＴＡＧＴＡＴＧＴＴＧＧＧＧＡＡＡＴＡＴＧＡＡＣＣＴＧＡＣＡＴＧＡＴＴＡＡＧＡＴＴＧＣＴＣＴＴＴＣＧＧＴＧＧＣＴＧＧ（配列番号３４）

First linker:
GTACCAGGCGCGCC (SEQ ID NO: 35)

p11:
TTTCATTTTGTTTTTTTCTATGCTATAAA (SEQ ID NO: 36)

GFP:
ＡＴＧＧＴＧＡＧＣＡＡＧＧＧＣＧＡＧＧＡＧＣＴＧＴＴＣＡＣＣＧＧＧＧＴＧＧＴＧＣＣＣＡＴＣＣＴＧＧＴＣＧＡＧＣＴＧＧＡＣＧＧＣＧＡＣＧＴＡＡＡＣＧＧＣＣＡＣＡＡＧＴＴＣＡＧＣＧＴＧＴＣＣＧＧＣＧＡＧＧＧＣＧＡＧＧＧＣＧＡＴＧＣＣＡＣＣＴＡＣＧＧＣＡＡＧＣＴＧＡＣＣＣＴＧＡＡＧＴＴＣＡＴＣＴＧＣＡＣＣＡＣＣＧＧＣＡＡＧＣＴＧＣＣＣＧＴＧＣＣＣＴＧＧＣＣＣＡＣＣＣＴＣＧＴＧＡＣＣＡＣＣＣＴＧＡＣＣＴＡＣＧＧＣＧＴＧＣＡＧＴＧＣＴＴＣＡＧＣＣＧＣＴＡＣＣＣＣＧＡＣＣＡＣＡＴＧＡＡＧＣＡＧＣＡＣＧＡＣＴＴＣＴＴＣＡＡＧＴＣＣＧＣＣＡＴＧＣＣＣＧＡＡＧＧＣＴＡＣＧＴＣＣＡＧＧＡＧＣＧＣＡＣＣＡＴＣＴＴＣＴＴＣＡＡＧＧＡＣＧＡＣＧＧＣＡＡＣＴＡＣＡＡＧＡＣＣＣＧＣＧＣＣＧＡＧＧＴＧＡＡＧＴＴＣＧＡＧＧＧＣＧＡＣＡＣＣＣＴＧＧＴＧＡＡＣＣＧＣＡＴＣＧＡＧＣＴＧＡＡＧＧＧＣＡＴＣＧＡＣＴＴＣＡＡＧＧＡＧＧＡＣＧＧＣＡＡＣＡＴＣＣＴＧＧＧＧＣＡＣＡＡＧＣＴＧＧＡＧＴＡＣＡＡＣＴＡＣＡＡＣＡＧＣＣＡＣＡＡＣＧＴＣＴＡＴＡＴＣＡＴＧＧＣＣＧＡＣＡＡＧＣＡＧＡＡＧＡＡＣＧＧＣＡＴＣＡＡＧＧＴＧＡＡＣＴＴＣＡＡＧＡＴＣＣＧＣＣＡＣＡＡＣＡＴＣＧＡＧＧＡＣＧＧＣＡＧＣＧＴＧＣＡＧＣＴＣＧＣＣＧＡＣＣＡＣＴＡＣＣＡＧＣＡＧＡＡＣＡＣＣＣＣＣＡＴＣＧＧＣＧＡＣＧＧＣＣＣＣＧＴＧＣＴＧＣＴＧＣＣＣＧＡＣＡＡＣＣＡＣＴＡＣＣＴＧＡＧＣＡＣＣＣＡＧＴＣＣＧＣＣＣＴＧＡＧＣＡＡＡＧＡＣＣＣＣＡＡＣＧＡＧＡＡＧＣＧＣＧＡＴＣＡＣＡＴＧＧＴＣＣＴＧＣＴＧＧＡＧＴＴＣＧＴＧＡＣＣＧＣＣＧＣＣＧＧＧＡＴＣＡＣＴＣＴＣＧＧＣＡＴＧＧＡＣＧＡＧＣＴＧＴＡＣＡＡＧＴＡＡ（配列番号３７）

Second linker:
GAGCTCCGGCCCGCTCGAGGCCGCTGGTACCCAACCT (SEQ ID NO: 38)

MH5 promoter:
AAAAATTGAAAATAAAATACAAAGGTTCTTTGAGGGTTGTGTTAAATTGAAAAGCGAGAAATAATCATAAATA (SEQ ID NO: 39)

Third Linker:
AGCCCGGT

Kozak array:
GCCACCATGG (SEQ ID NO:41) The 3′ end of the Kozak sequence overlaps with the 5′ end four nucleic acids of SEQ ID NO:29.

Nucleoprotein (SEQ ID NO: 29)

Fourth Linker:
GACCTGGAAGGCCCTAGATTCGAG (SEQ ID NO: 42)

Flag tag:
GACTACAAGGACGATGACGACAAG (SEQ ID NO: 43)

Stop codon:
TGA

Fifth Linker:
CTCGACCTGCAGTTTTTATG (SEQ ID NO: 44)

DelIII right flanking region:
ＧＡＡＡＧＴＴＴＴＡＴＡＧＧＴＡＧＴＴＧＡＴＡＧＡＡＣＡＡＡＡＴＡＣＡＴＡＡＴＴＴＴＧＴＡＡＡＡＡＴＡＡＡＴＣＡＣＴＴＴＴＴＡＴＡＣＴＡＡＴＡＴＧＡＣＡＣＧＡＴＴＡＣＣＡＡＴＡＣＴＴＴＴＧＴＴＡＣＴＡＡＴＡＴＣＡＴＴＡＧＴＡＴＡＣＧＣＴＡＣＡＣＣＴＴＴＴＣＣＴＣＡＧＡＣＡＴＣＴＡＡＡＡＡＡＡＴＡＧＧＴＧＡＴＧＡＴＧＣＡＡＣＴＴＴＡＴＣＡＴＧＴＡＡＴＣＧＡＡＡＴＡＡＴＡＣＡＡＡＴＧＡＣＴＡＣＧＴＴＧＴＴＡＴＧＡＧＴＧＣＴＴＧＧＴＡＴＡＡＧＧＡＧＣＣＣＡＡＴＴＣＣＡＴＴＡＴＴＣＴＴＴＴＡＧＣＴＧＣＴＡＡＡＡＧＣＧＡＣＧＴＣＴＴＧＴＡＴＴＴＴＧＡＴＡＡＴＴＡＴＡＣＣＡＡＧＧＡＴＡＡＡＡＴＡＴＣＴＴＡＣＧＡＣＴＣＴＣＣＡＴＡＣＧＡＴＧＡＴＣＴＡＧＴＴＡＣＡＡＣＴＡＴＣＡＣＡＡＴＴＡＡＡＴＣＡＴＴＧＡＣＴＧＣＴＡＧＡＧＡＴＧＣＣＧＧＴＡＣＴＴＡＴＧＴＡＴＧＴＧＣＡＴＴＣＴＴＴＡＴＧＡＣＡＴＣＧＣＣＴＡＣＡＡＡＴＧＡＣＡＣＴＧＡＴＡＡＡＧＴＡＧＡＴＴＡＴＧＡＡＧＡＡＴＡＣＴＣＣＡＣＡＧＡＧＴＴＧＡＴＴＧＴＡＡＡＴＡＣＡＧＡＴＡＧＴＧＡＡＴＣＧＡＣＴＡＴＡＧＡＣＡＴＡＡＴＡＣＴＡＴＣＴＧＧＡＴＣＴＡＣＡＣＡＴＴＣＡＣＣＧＧＡＡＡＣＴＡＧＴＴＧ（配列番号４５）

Plasmid DNA was purified from transformed bacteria (E. coli K12 DH10B™ T1R) and concentrations were determined by UV spectroscopy with GeneArt (Thermofisher).

BHK-21 cells were infected with MVA 1974 at a multiplicity of infection of 0.05. Infected cells were transfected with pMVAHantaNP using Lipofectamine (Life Technologies) as directed by the manufacturer. The resulting recombinant MVA HantaNP was serially plaque-purified four times in chicken embryo fibroblast (“CEF”) cells based on GFP expression. MVA HantaNP was amplified on CEF cells, purified by sucrose cushion centrifugation, and titrated by plaque assay on CEF cells prior to in vivo use. Plaques were visualized by immunostaining with rabbit anti-vaccinia antibody (AbD Serotec, UK) and Vectastain Universal ABC-AP kit (Vector laboratories, USA) using GFP fluorescence. Genomic DNA was extracted from infected cells using the Wizard SV genomic DNA purification system (Promega, USA) and used as template in PCR using the KAPA2G Fast HotStart PCR kit (KAPABiosystems, USA) for genotyping. .

The presence of the MVAHantaNP construct was confirmed by polymerase chain reaction (PCR). A set of primers was specifically designed to check the expected size of 3260 bp from the Hanta NP to the MVA flanking region (shown in Figure 2).

Sequencing of the expressed protein confirmed very high sequence fidelity. Recombinant purified MVA HantaNP was then expanded stepwise by tissue culture in increasing flask sizes. MVA HantaNPs were first grown in chicken embryo fibroblast (CEF) cells in small flasks, harvested and then infected into CEF cells in slightly larger flasks. This process was repeated into increasingly larger flasks until MVA HantaNP successfully infected CEF cells in 10-fold larger flasks. Sucrose cushion centrifugation was performed and virus pellets were resuspended in PBS ready for immunogenicity testing. A total of 6 batches were prepared. Batches 2+3 and 4+5+6 were pooled into a single sample and titrated for virus concentration.

Purified vaccine batches are aligned with a positive control (original plasmid obtained from Geneart). A second set of primers was designed to identify the entire insert from both MVA flanking regions. The results show the presence of pure recombinant MVA (MVA with insert) in all vaccine batches. Again, the original plasmid was used as a positive control and all vaccine batches have the same expected size product as the positive control.

Details of the primers are as follows:
SEQ ID NO: 46: CGGCACCTCTCTTAAGAAGT (Fwd, targeting Del III left flanking region)
SEQ ID NO: 47: GTGTAGCGTATACTAATGATATTAG (targets Rev, Del III right flanking region)
SEQ ID NO: 48: GGAGTACAACTACAACAGCCACAACG (Fwd, targeting GFP)

The GFP Fwd primer binds to the GFP sequence and when used in combination with the Rev Del III right flanking primer extends from GFP through the nuclear protein to the right MVA flanking region and specifically identifies the presence of the NP gene.

Detection of Protein Expression CEF cells were infected with MVA HantaNP at a multiplicity of infection of 0.05 and incubated at 37° C. in modified Eagle's medium (MEM) supplemented with 2% FBS (Sigma-Aldrich. UK). Medium was removed after 48 hours and good GFP fluorescence and CPE were observed microscopically. Cells were lysed with 1× LDS Nupage® Reducing Sample Buffer (Nupage® LDS Sample Buffer containing 1× Nupage® Sample Reducing Buffer) (Thermofisher, UK) and placed in an Eppendorf tube. and heated at 70° C. for 10 minutes. Uninfected cells were treated in the same way as negative controls. MVA HantaNP lysates were subjected to SDS-PAGE on 4-12% Bis-Tris gels (Life technologies) and proteins were transferred to nitrocellulose membranes. Nitrocellulose membranes were blocked using 5% milk powder (Merck Millipore) and then 1-2 in the presence of primary antibody (rabbit anti-V5 polyclonal (Invitrogen) at 1/1000 in PBS-0.05% Tween). After an hour rocking incubation, the cells were washed three times with PBS containing 0.05% Tween-20 (Sigma-Aldrich). Membranes were incubated in the presence of HRP-conjugated secondary antibody (1/1000 anti-rabbit IgG peroxidase (Sigma-Aldrich) in PBS-0.05% Tween) for 1 hour with rocking and washed as before. . Protein expression was determined by detection of bound antibody using the Pierce ECL WB substrate kit (Thermofisher) according to the manufacturer's instructions and visualized in a Chemi-Illuminant Imager (Syngene). Molecular weights were determined using the molecular ladder MagicMark XP Western Protein Standard (Invitrogen) as a reference.

Western blot analysis (see Figure 3) confirmed the expression of the flag tag located downstream of the NP. The predicted size of the protein (NP+linker and flag tag) is 89 kDa and the protein sequence is provided in SEQ ID NO:49. Expression is observed from the passage 3 harvest through to the vaccine batch (we observed low levels of proteolysis, but this is not considered significant). The band of interest is located at the expected size of the protein, again suggesting good expression.
ＭＡＴＭＥＥＩＱＲＥＩＳＡＨＥＧＱＬＶＩＡＲＱＫＶＫＤＡＥＫＱＹＥＫＤＰＤＤＬＮＫＲＡＬＨＤＲＥＳＶＡＡＳＩＱＳＫＩＤＥＬＫＲＱＬＡＤＲＩＡＡＧＫＮＩＧＱＤＲＤＰＴＧＶＥＰＧＤＨＬＫＥＲＳＡＬＳＹＧＮＴＬＤＬＮＳＬＤＩＤＥＰＴＧＱＴＡＤＷＬＴＩＩＶＹＬＴＳＦＶＶＰＩＩＬＫＡＬＹＭＬＴＴＲＧＲＱＴＳＫＤＮＫＧＭＲＩＲＦＫＤＤＳＳＹＥＤＶＮＧＩＲＫＰＫＨＬＹＶＳＭＰＮＡＱＳＳＭＫＡＥＥＩＴＰＧＲＦＲＴＡＶＣＧＬＹＰＡＱＩＫＡＲＮＭＶＳＰＶＭＳＶＶＧＦＬＡＬＡＫＤＷＴＳＲＩＥＥＷＬＧＡＰＣＫＦＭＡＥＳＰＩＡＧＳＬＳＧＮＰＶＮＲＤＹＩＲＱＲＱＧＡＬＡＧＭＥＰＫＥＦＱＡＬＲＱＨＳＫＤＡＧＣＴＬＶＥＨＩＥＳＰＳＳＩＷＶＦＡＧＡＰＤＲＣＰＰＴＣＬＦＶＧＧＭＡＥＬＧＡＦＦＳＩＬＱＤＭＲＮＴＩＭＡＳＫＴＶＧＴＡＤＥＫＬＲＫＫＳＳＦＹＱＳＹＬＲＲＴＱＳＭＧＩＱＬＤＱＲＩＩＶＭＦＭＶＡＷＧＫＥＡＶＤＮＦＨＬＧＤＤＭＤＰＥＬＲＳＬＡＱＩＬＩＤＱＫＶＫＥＩＳＮＱＥＰＭＫＬＭＬＳＹＧＮＶＬＤＬＮＨＬＤＩＤＥＰＴＧＱＴＡＤＷＬＧＩＶＩＹＬＴＳＦＶＶＰＩＬＬＫＡＬＹＭＬＴＴＲＧＲＱＴＴＫＤＮＫＧＴＲＩＲＦＫＤＤＳＳＦＥＤＶＮＧＩＲＫＰＫＨＬＹＶＳＬＰＮＡＱＳＳＭＫＡＥＥＩＴＰＧＲＹＲＴＡＩＣＧＬＹＰＡＱＩＫＡＲＱＭＩＳＰＶＭＳＶＩＧＦＬＡＬＡＫＤＷＳＤＲＩＥＱＷＬＳＥＰＣＫＬＬＰＤＴＡＡＶＳＬＬＧＧＰＡＴＮＲＤＹＬＲＱＲＱＶＡＬＧＮＭＥＴＫＥＳＫＡＩＲＱＨＡＥＡＡＧＣＳＭＩＥＤＩＥＳＰＳＳＩＷＶＦＡＧＡＰＤＲＣＰＰＴＣＬＦＩＡＧＭＡＥＬＧＡＦＦＳＩＬＱＤＭＲＮＴＩＭＡＳＫＴＶＧＴＳＥＥＫＬＲＫＫＳＳＦＹＱＳＹＬＲＲＴＱＳＭＧＩＱＬＤＱＲＩＩＶＬＦＭＶＡＷＧＫＥＡＶＤＮＦＨＬＧＤＤＭＤＰＥＬＲＴＬＡＱＳＬＩＤＶＫＶＫＥＩＳＮＱＥＰＬＫＬＤＬＥＧＰＲＦＥＤＹＫＤＤＤＤＫ（配列番号４９）

The amino acid sequence of SEQ ID NO:49 corresponds to the amino acid sequence of SEQ ID NO:31 plus the expressed fourth linker and flag tag.

Example 2
Immunogenicity of MVA HantaNP in A129 Mice Eighty male 6-8 week old A129 mice were randomized into four groups, ear-tagged and then vaccinated.

Group 1 received two dose vaccinations of MVA HantaNP in endotoxin-free phosphate-buffered saline (PBS) at 1×10 ⁷ pfu per animal on days 0 and 14.

Group 2 received a single vaccination on day 14 with MVA HantaNP in endotoxin-free PBS at 1×10 ⁷ plaque forming units (pfu) per animal.

Three groups received two dose vaccinations of MVA empty vector in endotoxin-free PBS on days 0 and 14 at 1×10 ⁷ pfu per animal.

Group 4 received two dose vaccinations of endotoxin-free PBS on days 0 and 14 as negative controls.

All mice were injected intramuscularly in the caudal thigh. 100 μl was administered at each vaccination (50 μl in each thigh). Animal weights were recorded daily throughout the study. Five animals from each group were euthanized and splenic tissue and blood were collected 28 days after primary vaccination. All efforts were made to minimize animal suffering. These studies were approved by the ethical review processes of PHE, Porton Down, UK and the UK Home Office via project license number 30/2993. Work was performed in accordance with the Animals (Scientific Procedures) Act, 1986 and the UK Home Office Code of Practice for the Care and Management of Animals Used in Scientific Procedures (1989).

No clinical signs were observed with vaccination throughout the study and all mice gained weight as expected (see Figure 4a). All four groups gained weight throughout the study, as expected, with group 4 having a consistently lower body weight than groups 1-3. By the end of the study, similar percent weight gain was observed in all groups. These clinical data demonstrate that mice tolerated the vaccine without adverse effects.

To determine T cell responses in immunized animals, an interferon-gamma ELISPOT assay was used to measure the frequency of responsive T cells after stimulation with hantavirus-specific peptides.

Spleens were aseptically harvested from test animals, homogenized, and red blood cells lysed. Splenocytes were resuspended in RPMI medium (Sigma-Aldrich) supplemented with 5% FBS, 2 mM L-glutamine, 100 U penicillin and 0.1 mg/ml streptomycin, 50 mM 2-mercaptoethanol and 25 mM HEPES solution (Sigma-Aldrich). muddy. Splenocytes were assessed for antigen recall responses by IFN-γ ELISPOT (Mabtech, Sweden) performed according to the manufacturer's instructions. Cells were seeded at 2×10e6 per well in PVDF microtiter plates and restimulated with peptide pools (JPT, Berlin).

The peptide span of the Hanta NP protein sequence was 15 residues long with 11 residues overlap between peptides. A total of 189 peptides were produced and tested as 11 peptide pools (see Table 1).
Table 1: Peptide pools (starting amino acid (“AA”) numbering corresponds to amino acid numbering in SEQ ID NO:31)

These were applied to the cells at a final concentration of 2.5 μg/ml per peptide, 17 peptides each for pools 1-10 and 19 peptides for pool 11. Plates were developed after 18 hours at 37° C., 5% CO ₂ in a humidified incubator. Spots were counted visually with an automated ELISPOT reader (Cellular Technologies Limited, USA). Background values from wells containing cells and medium but no peptide were subtracted and data presented as responses to individual pools or summed across target proteins. Results were expressed as spot forming units (SFU) per 10 ⁶ cells.

The MVA-WT and PBS groups (groups 3 and 4) were negative when stimulated with all Hanta NP pools. In the prime/boost and prime groups, IFN-γ responses were detected against several peptide pools, with particularly strong responses directed to two different regions of NP (corresponding to pools 4 and 9).

We found that T cell (IFN-γ) stimulation was greatly increased for SEQ ID NOs:11 and 12.

Increased responses were also detected for pools 2, 3, 5, 7, 8 and 10 for the prime/boost and prime groups compared to the control group. Total ELISPOT responses from vaccinated and non-vaccinated mice are shown in FIG. Figure 6 shows ELISPOT responses to individual peptide pools.

To measure antibody responses in immunized mice, an ELISA assay was performed to assess antibody binding to hantavirus-specific proteins. Recombinant Hanta NP (Native Antigen Company, UK) as a crude lysate was diluted in 0.2M carbonate-bicarbonate buffer pH 9.4 (Thermo Scientific) for coating Maxisorp 96-well plates (Nunc, Denmark). 100 μl at 10 μg/ml was used for . Plates were incubated overnight at 4°C, then washed with PBS + 0.01% Tween-20 (Sigma-Aldrich) and incubated at 37°C with 100 μl of 5% milk powder (Merck, Millipore) in PBS + 0.01% Tween-20. After blocking for time, it was washed again with PBS + 0.01% Tween-20. Samples were diluted 1:50 with 5% milk powder in PBS + 0.01% Tween-20 buffer, added to the plate in triplicate (100 µl/well) and incubated at 37°C for 1 hour. Normal mouse serum (Sigma-Aldrich) and polyclonal anti-hantavirus hyperimmune mouse ascetic fluid samples (BEI Resources, USA) were used as positive and negative control samples, respectively. Plates were washed with PBS + 0.01% Tween-20 and 100 μl of polyclonal anti-mouse-HRP conjugate (Sigma-Aldrich) diluted 1:20,000 in 5% milk PBS + 0.01% Tween-20 was added to each well. . After an additional 1 hour incubation at 37°C, the plates were washed with PBS + 0.01% Tween-20 and 100 µl of TMB substrate (Surmodics) was added to each well followed by incubation at 20°C for 1 hour. The reaction is stopped by adding 100 μl of stop solution (Surmodics) prepared according to the manufacturer's instructions and the plate is read at 450 nm using a molecular devices plate reader and Softmax Pro version 5.2 software (Molecular Devices). rice field. Background absorbance values were subtracted from sample values and results were reported as absorbance (450 nm) at 1:50 dilution. Data were plotted and analyzed using Graph Pad Prism 7 (see Figure 7).

The MVA-WT and PBS control groups showed very faint absorbance values similar to blank wells. Responses in all mice in both prime and prime/boost vaccination groups were significantly higher. The prime-only group recorded an average absorbance of about 2.3 and the prime/boost group recorded an average OD of about 1.5.

Thus, the vectors of the present invention demonstrate highly desirable induction of cellular and humoral immune responses.

Example 3
Efficacy study Sixty male A129 mice weighing 19-21 g were pre-randomized into four groups and then ear-tagged and microchipped for identification, weight monitoring and temperature monitoring.

The remaining mice not terminated on day 28 for immunogenicity testing were challenged on day 28 with Hanta SEOV. From each group, n=10 animals were challenged via the intranasal route and n=5 animals were challenged via the intramuscular route with 1.36×10 ⁶ TCID50/dose.

Animals challenged intramuscularly were euthanized on day 33. Intranasally challenged mice were euthanized on day 33 (5/group) or day 42 (5/group). Blood, saliva, liver, kidney, lung and spleen were collected for histology and viral load analysis. All efforts were made to minimize animal suffering. These studies were approved by the ethical review processes of PHE, Porton Down, UK and the UK Home Office via project license number 30/2993. Work was performed in accordance with the Animals (Scientific Procedures) Act, 1986 and the UK Home Office Code of Practice for the Care and Management of Animals Used in Scientific Procedures (1989).

Clinical signs:
Animal weights and temperatures were recorded daily throughout the study. All challenged animals remained healthy and no clinical signs were observed after challenge with hantavirus. Body temperature and body weight throughout the study are reported in FIG.

Viral load:
Viral load was assessed 5 and 14 days after challenge. As shown in Figure 9, on day 5, immunization with MVAHantaNP achieved a reduction or complete clearance of hantavirus from the tissues tested. A highly favorable reduction in viral load was also observed in most tissues 14 days after challenge.

Viral Load - Follow-up:
In a follow-up study, 28 female A129 mice were pre-randomized into two groups, followed by ear tagging and microchipping for identification, weight monitoring and temperature monitoring.

Of these 28 mice, 16 were primed with GLP-grade MVA HantaNPs on day 0 followed by a boost immunization on day 14 ("Group A"), and 12 mice were given an empty MVA wild Prime and boost immunizations were given with type vector on days 0 and 14, respectively (“Group B”). Immunizations were performed according to Example 2 above.

On day 28, 8 mice in Group A and 8 mice in Group B were challenged intranasally with Hanta SEOV at a dose of 3×10 ⁶ TCID50/mouse.

In this follow-up, viral load was assessed 5 days after challenge. As shown in Figure 10, immunization with MVA HantaNP achieved a beneficial reduction of hantavirus from the tissues tested, even when the challenge dose was more than doubled.

Example 4
Preparation of Adenoviral Vectors of the Examples Non-replicating adenoviruses are engineered to express the hantavirus NP nucleic acids of the invention or fragments thereof. The hantavirus NP gene sequence is inserted into the adenovirus vector genome. Hantavirus NP expression is demonstrated by reactivity between NP-specific antibodies and adenovirus-derived products by Western blotting or ELISA as follows:

Cell lysates of recombinant adenovirus-infected cells subjected to SDS-PAGE and western blotting with antibodies specific for hantavirus NP show specific reactivity compared to negative controls.

Alternatively, products from cells infected with recombinant adenovirus are used to coat ELISA plates. Hantavirus-specific antibodies bind to the coating and are detected via a chemical reaction.

Example 5
Hantavirus Vaccines Provide Cross-Strain Protection Vaccines expressing the hantavirus NP nucleic acids of the invention or fragments thereof in adenovirus or non-replicating poxvirus vectors are administered to mice susceptible to disease caused by hantavirus by the parenteral route. deliver through They are challenged with a lethal dose of hantavirus from a strain other than the strain on which the vaccine is based. Challenged animals show no or mild clinical signs of illness and do not require euthanasia. Control animals given the same challenge dose of hantavirus but not vaccinated show severe signs of illness, reach humane clinical endpoints, and require euthanasia.

Example 6
Preparation and Validation of Recombinant Influenza Virus Vectors Reverse genetics is used to construct recombinant influenza viruses that carry the protective epitopes of hantavirus NP in their neuraminidase stalks. Hantavirus-specific cytotoxic T lymphocytes (CTL) are induced in mice after intranasal or parenteral administration. These CTLs lead to a reduction in viral load and clinical illness after Hantavirus challenge.

Example 7
Preparation and Efficacy of Recombinant Bacterial Vectors Hantavirus NP nucleic acids or fragments thereof of the invention are expressed on the surface of genetically attenuated Gram-negative bacteria. Following intranasal or parenteral administration to mice, bacterial vectors colonize antigen-presenting cells (eg, dendritic cells or macrophages). Humoral and cellular hantavirus-specific immune responses are induced. These immune responses result in a reduction in viral load and clinical illness after Hantavirus challenge.

Claims (45)

A viral or bacterial vector comprising a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof and capable of inducing an immune response in a subject. 2. A nucleic acid sequence according to claim 1, wherein the nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NOS: 1, 2 and 3. vector. 3. A nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 22, 23 or 24. vector. 4. Any one of claims 1 to 3, wherein the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 15, 16 or 17. The vector described in section. 2. from claim 1, wherein the nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NOS: 5, 6, 8 and 9 5. The vector according to any one of 4. 6. Any of claims 1-5, wherein the nucleic acid sequence encoding the hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 25, 26, 27 or 28. or the vector according to item 1. 7. Any of claims 1-6, wherein the nucleic acid sequence encoding the hantavirus nucleoprotein or antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 18, 19, 20 or 21. or the vector according to item 1. a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof comprising a first nucleic acid sequence and a second nucleic acid sequence;
(A) said first nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 15, 16, 17, 22, 23 or 24; and (B) said second the nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 18, 19, 20, 21, 25, 26, 27 or 28;
8. The vector according to any one of claims 1-7. a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof comprising a first nucleic acid sequence and a second nucleic acid sequence;
(A) said first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:24; and (B) said second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:28. having % sequence identity,
9. A vector according to any one of claims 1-8. a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof comprising a first nucleic acid sequence and a second nucleic acid sequence;
(A) said first nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 22 or 23; and (B) said second nucleic acid sequence is SEQ ID NO: 25, provided by a nucleic acid sequence having at least 70% sequence identity with a nucleic acid sequence selected from 26 or 27;
10. A vector according to any one of claims 1-9. a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof comprising a first nucleic acid sequence and a second nucleic acid sequence;
(A) said first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 17; and (B) said second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO: 21. having % sequence identity,
A vector according to any one of claims 1 to 10. a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof comprising a first nucleic acid sequence and a second nucleic acid sequence;
(A) said first nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 15 or 16; and (B) said second nucleic acid sequence is SEQ ID NO: 18, having at least 70% sequence identity with a nucleic acid sequence selected from 19 or 20;
A vector according to any one of claims 1 to 11. a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof comprising a first nucleic acid sequence and a second nucleic acid sequence;
(A) said first nucleic acid sequence has at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 1, 2 or 3; and (B) said second nucleic acid sequence has SEQ ID NO: having at least 70% sequence identity with a nucleic acid sequence selected from 5, 6, 8 or 9;
13. A vector according to any one of claims 1-12. a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof comprising a first nucleic acid sequence and a second nucleic acid sequence;
(A) said first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:3; and (B) said second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:9. having % sequence identity,
14. A vector according to any one of claims 1-13. a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof comprising a first nucleic acid sequence and a second nucleic acid sequence;
(A) said first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:2; and (B) said second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:8. having % sequence identity,
15. A vector according to any one of claims 1-14. a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof comprising a first nucleic acid sequence and a second nucleic acid sequence;
(A) said first nucleic acid has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:2; and (B) said second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:6. having the sequence identity of
16. A vector according to any one of claims 1-15. a nucleic acid sequence encoding a hantavirus nucleoprotein or an antigenic fragment thereof comprising a first nucleic acid sequence and a second nucleic acid sequence;
(A) said first nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:1; and (B) said second nucleic acid sequence has at least 70% sequence identity with the nucleic acid sequence of SEQ ID NO:5. having % sequence identity,
17. A vector according to any one of claims 1-16. 18. The vector of any one of claims 1-17, wherein the nucleic acid sequence encoding the hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO:29. 18. The vector of any one of claims 1-17, wherein the nucleic acid sequence encoding the hantavirus nucleoprotein or an antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO:30. 20. The vector of any one of claims 1-19, which is a viral vector. 21. The vector of claim 20, which is a non-replicating poxvirus vector. 22. The vector of claim 21, wherein the non-replicating poxvirus vector is selected from modified vaccinia virus Ankara (MVA) vectors, NYVAC vaccinia virus vectors, canarypox (ALVAC) vectors, and fowlpox (FPV) vectors. 23. The vector of claim 21 or claim 22, wherein said non-replicating poxvirus vector is an MVA vector. 23. The vector of claim 21 or claim 22, wherein said non-replicating poxvirus vector is a fowlpox vector. 21. The vector of claim 20, which is an adenoviral vector. 26. The vector of claim 25, wherein said adenoviral vector is a non-replicating adenoviral vector. The adenoviral vector is a human adenoviral vector, a simian adenoviral vector, a group B adenoviral vector, a group C adenoviral vector, a group E adenoviral vector, an adenoviral 6 vector, a PanAd3 vector, an adenoviral C3 vector, a ChAdY25 vector, 27. The vector of claim 25 or 26, selected from AdC68 vectors and Ad5 vectors. 21. The vector of claim 20, which is a measles virus vector. 29. The vector of claim 28, wherein said measles virus vector is a non-replicating measles virus vector. 30. The vector of any of claims 1-29, wherein the hantavirus nucleoprotein comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 4, or an antigenic fragment thereof. 31. The vector of claim 30, wherein said antigenic fragment comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:13. 32. The vector of claim 30 or 31, wherein said antigenic fragment comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:11. 33. Any of claims 1-32, wherein the hantavirus nucleoprotein comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NOS: 7 and 10, or an antigenic fragment thereof. vector. 34. The vector of claim 33, wherein said antigenic fragment comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:14. 35. The vector of any one of claims 30-34, wherein said antigenic fragment comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:12. 36. A nucleic acid sequence encoding a viral vector according to any one of claims 1-35. A method of preparing a viral vector, comprising:
providing a nucleic acid comprising a nucleic acid sequence encoding a vector according to any one of claims 1-35;
transfecting the nucleic acid into a host cell;
culturing the host cell under conditions suitable for the propagation of the vector;
obtaining said vector from said host cell;
method including. 37. A host cell comprising the nucleic acid sequence of claim 36. 36. A composition comprising the vector of any one of claims 1-35 and a pharmaceutically acceptable carrier. 40. The composition of Claim 39, further comprising an adjuvant. 41. A vector according to any one of claims 1 to 35, or a composition according to claim 39 or claim 40 for use in medicine. 36. A vector according to any one of claims 1 to 35, or a composition according to claim 27 or claim 28 for use in a method of inducing an immune response in a subject. 43. Vector for use according to claim 42, wherein said immune response comprises a T cell response. 41. A vector according to any one of claims 1 to 35, or a composition according to claim 39 or claim 40 for use in a method of preventing or treating hantavirus infection in a subject. 45. The vector of claim 44 or composition of claim 44 for use in a method of preventing or treating renal symptomatic hemorrhagic fever in a subject.

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